Endocrine Involvement in Developmental Syndromes (Endocrine Development Vol 14)

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Author: Marco Cappa | Mohamad Maghnie | Sandro Loche | Gian Franco Bottazzo

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Endocrine Involvement in Developmental Syndromes

This book has been printed with financial support from Pfizer Italia

Endocrine Development Vol. 14

Series Editor

P.-E. Mullis

Bern

Workshop, April 21–22, 2008, Rome

Endocrine Involvement in Developmental Syndromes Volume Editors

Marco Cappa Rome Mohamad Maghnie Genova Sandro Loche Cagliari Gian Franco Bottazzo Rome 26 figures, 2 in color, and 15 tables, 2009

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney

Endocrine Development Founded 1999 by Martin O. Savage, London

Marco Cappa

Mohamad Maghnie

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Department of Pediatrics IRCCS G. Gaslini University of Genova Genova, Italy

Sandro Loche

Gian Franco Bottazzo

Regional Hospital for Microcytaemia Cagliari, Italy

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Library of Congress Cataloging-in-Publication Data Workshop on Endocrine Involvement in Developmental Syndromes (2008 : Rome, Italy) Endocrine involvement in developmental syndromes / Workshop on Endocrine Involvement in Developmental Syndromes, April 21-22, 2008, Rome ; volume editors, Marco Cappa ... [et al.]. p. ; cm. -- (Endocrine development, ISSN 1421-7082 ; v. 14) Includes bibliographical references and indexes. ISBN 978-3-8055-9041-9 (hard cover : alk. paper) 1. Pediatric endocrinology--Congresses. 2. Growth disorders--Endocrine aspects--Congresses. 3. Developmental disabilities--Endocrine aspects--Congresses. I. Cappa, Marco. II. Title. III. Series: Endocrine development ; v. 14. [DNLM: 1. Endocrine System Diseases--etiology--Congresses. 2. Congenital Abnormalities--Congresses. 3. Genetic Diseases, Inborn--complications--Congresses. W1 EN3635 v.14 2009 / WK 140 W926e 2009] RJ482.G76.W67 2009 618.92'4--dc22 2008052202 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 978–3–8055–9041–9

Contents

VII Preface Cappa, M.; Bottazzo, G.F. (Rome) 1

10 20 29

38 53

61 67

83 95

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in BeckwithWiedemann Syndrome and Wilms’ Tumor Riccio, A. (Caserta/Naples); Sparago, A.; Verde, G. (Naples); De Crescenzo, A. (Caserta); Citro, V.; Cubellis, M.V. (Naples); Ferrero, G.B.; Silengo, M.C. (Torino); Russo, S.; Larizza, L. (Milan); Cerrato, F. (Caserta) Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Eggermann, T. (Aachen) Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Gurrieri, F.; Accadia, M. (Rome) Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches Barberi, L.; Dobrowolny, G.; Pelosi, L.; Giacinti, C.; Musarò, A. (Rome) Mitochondrial Encephalomyopathies and Related Syndromes: Brief Review Bertini, E.; D’Amico A. (Rome) Overgrowth Syndromes: A Classification Neri, G.; Moscarda, M. (Rome) C-Type Natriuretic Peptide and Overgrowth Bocciardi, R.; Ravazzolo, R. (Genova) Role of Transcription Factors in Midline Central Nervous System and Pituitary Defects Kelberman, D.; Dattani, M.T. (London) Developmental Abnormalities of the Posterior Pituitary Gland di Iorgi, N.; Secco, A.; Napoli, F.; Calandra, E.; Rossi, A.; Maghnie, M. (Genova) Hyperinsulinism in Developmental Syndromes Kapoor, R.R.; James, C.; Hussain, K. (London)

114

135 143

151 167

174

181 182

VI

Developmental Syndromes: Growth Hormone Deficiency and Treatment Mazzanti, L.; Tamburrino, F.; Bergamaschi, R.; Scarano, E.; Montanari, F.; Torella, M.; Ballarini, E.; Cicognani, A. (Bologna) Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Chernausek, S.D. (Oklahoma City, Okla.) Phenotypic Aspects of Growth Hormone- and IGF-I-Resistant Syndromes Savage, M.O.; David, A.; Camacho-Hübner, C.; Metherell, L.A.; Clark, A.J.L. (London/Stockholm) Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Pozzilli, P.; Guglielmi, C. (Rome) Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection Main, K.M.; Skakkebæk, N.E. (Copenhagen); Toppari, J. (Turku) Disorders of Sex Development in Developmental Syndromes Hiort, O.; Gillessen-Kaesbach, G. (Lübeck) Author Index Subject Index

Contents

Preface

Recent years have seen a significant improvement in the knowledge of genetics and developmental syndromes. In this scenario, the study of endocrinological aspects in patients with genetic syndromes acquires increasing interest and significance. The workshop on “Endocrine Involvement in Developmental Syndromes”, held in Rome on April 21–22, 2008, represented a precious opportunity to provide an updated global view of this important field of medical sciences. The scientific program of the workshop included the most recent advances in the study of developmental syndromes and epigenetics, with world-wide experts focusing their contributions on modern concepts of basic and clinical science in order to clarify genetic, clinical and biological aspects of these syndromes. This book is the result of this fruitful exchange of experience and knowledge, and it provides a thorough elucidation of endocrine involvement and epigenetic aspects of various developmental syndromes, opening new ways to manage the complexity of such a topic. We are confident that the state-of-art information provided by this book will be of great interest for endocrinologists, pediatricians, and genetists involved in the study and treatment of developmental syndromes. Marco Cappa Gian Franco Bottazzo Rome

Endocrine Involvement in Developmental Syndromes

This book has been printed with financial support from Pfizer Italia

Endocrine Development Vol. 14

Series Editor

P.-E. Mullis

Bern

Workshop, April 21–22, 2008, Rome

Endocrine Involvement in Developmental Syndromes Volume Editors

Marco Cappa Rome Mohamad Maghnie Genova Sandro Loche Cagliari Gian Franco Bottazzo Rome 26 figures, 2 in color, and 15 tables, 2009

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Shanghai · Singapore · Tokyo · Sydney

Endocrine Development Founded 1999 by Martin O. Savage, London

Marco Cappa

Mohamad Maghnie

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Department of Pediatrics IRCCS G. Gaslini University of Genova Genova, Italy

Sandro Loche

Gian Franco Bottazzo

Regional Hospital for Microcytaemia Cagliari, Italy

Department of Pediatrics Pediatric Hospital Bambino Gesù Rome, Italy

Library of Congress Cataloging-in-Publication Data Workshop on Endocrine Involvement in Developmental Syndromes (2008 : Rome, Italy) Endocrine involvement in developmental syndromes / Workshop on Endocrine Involvement in Developmental Syndromes, April 21-22, 2008, Rome ; volume editors, Marco Cappa ... [et al.]. p. ; cm. -- (Endocrine development, ISSN 1421-7082 ; v. 14) Includes bibliographical references and indexes. ISBN 978-3-8055-9041-9 (hard cover : alk. paper) 1. Pediatric endocrinology--Congresses. 2. Growth disorders--Endocrine aspects--Congresses. 3. Developmental disabilities--Endocrine aspects--Congresses. I. Cappa, Marco. II. Title. III. Series: Endocrine development ; v. 14. [DNLM: 1. Endocrine System Diseases--etiology--Congresses. 2. Congenital Abnormalities--Congresses. 3. Genetic Diseases, Inborn--complications--Congresses. W1 EN3635 v.14 2009 / WK 140 W926e 2009] RJ482.G76.W67 2009 618.92'4--dc22 2008052202 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® Disclaimer. The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in the book is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2009 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free and non-aging paper (ISO 9706) by Reinhardt Druck, Basel ISSN 1421–7082 ISBN 978–3–8055–9041–9

Contents

VII Preface Cappa, M.; Bottazzo, G.F. (Rome) 1

10 20 29

38 53

61 67

83 95

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in BeckwithWiedemann Syndrome and Wilms’ Tumor Riccio, A. (Caserta/Naples); Sparago, A.; Verde, G. (Naples); De Crescenzo, A. (Caserta); Citro, V.; Cubellis, M.V. (Naples); Ferrero, G.B.; Silengo, M.C. (Torino); Russo, S.; Larizza, L. (Milan); Cerrato, F. (Caserta) Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Eggermann, T. (Aachen) Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Gurrieri, F.; Accadia, M. (Rome) Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches Barberi, L.; Dobrowolny, G.; Pelosi, L.; Giacinti, C.; Musarò, A. (Rome) Mitochondrial Encephalomyopathies and Related Syndromes: Brief Review Bertini, E.; D’Amico A. (Rome) Overgrowth Syndromes: A Classification Neri, G.; Moscarda, M. (Rome) C-Type Natriuretic Peptide and Overgrowth Bocciardi, R.; Ravazzolo, R. (Genova) Role of Transcription Factors in Midline Central Nervous System and Pituitary Defects Kelberman, D.; Dattani, M.T. (London) Developmental Abnormalities of the Posterior Pituitary Gland di Iorgi, N.; Secco, A.; Napoli, F.; Calandra, E.; Rossi, A.; Maghnie, M. (Genova) Hyperinsulinism in Developmental Syndromes Kapoor, R.R.; James, C.; Hussain, K. (London)

114

135 143

151 167

174

181 182

VI

Developmental Syndromes: Growth Hormone Deficiency and Treatment Mazzanti, L.; Tamburrino, F.; Bergamaschi, R.; Scarano, E.; Montanari, F.; Torella, M.; Ballarini, E.; Cicognani, A. (Bologna) Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Chernausek, S.D. (Oklahoma City, Okla.) Phenotypic Aspects of Growth Hormone- and IGF-I-Resistant Syndromes Savage, M.O.; David, A.; Camacho-Hübner, C.; Metherell, L.A.; Clark, A.J.L. (London/Stockholm) Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Pozzilli, P.; Guglielmi, C. (Rome) Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection Main, K.M.; Skakkebæk, N.E. (Copenhagen); Toppari, J. (Turku) Disorders of Sex Development in Developmental Syndromes Hiort, O.; Gillessen-Kaesbach, G. (Lübeck) Author Index Subject Index

Contents

Preface

Recent years have seen a significant improvement in the knowledge of genetics and developmental syndromes. In this scenario, the study of endocrinological aspects in patients with genetic syndromes acquires increasing interest and significance. The workshop on “Endocrine Involvement in Developmental Syndromes”, held in Rome on April 21–22, 2008, represented a precious opportunity to provide an updated global view of this important field of medical sciences. The scientific program of the workshop included the most recent advances in the study of developmental syndromes and epigenetics, with world-wide experts focusing their contributions on modern concepts of basic and clinical science in order to clarify genetic, clinical and biological aspects of these syndromes. This book is the result of this fruitful exchange of experience and knowledge, and it provides a thorough elucidation of endocrine involvement and epigenetic aspects of various developmental syndromes, opening new ways to manage the complexity of such a topic. We are confident that the state-of-art information provided by this book will be of great interest for endocrinologists, pediatricians, and genetists involved in the study and treatment of developmental syndromes. Marco Cappa Gian Franco Bottazzo Rome

Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 1–9

Inherited and Sporadic Epimutations at the IGF2-H19 Locus in Beckwith-Wiedemann Syndrome and Wilms’ Tumor Andrea Riccioa,b ⭈ Angela Sparagob ⭈ Gaetano Verdeb ⭈ Agostina De Crescenzoa ⭈ Valentina Citroc ⭈ Maria Vittoria Cubellisc ⭈ Giovanni Battista Ferrerod ⭈ Margherita Cirillo Silengod ⭈ Silvia Russoe ⭈ Lidia Larizzae,f ⭈ Flavia Cerratoa a

Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta; bIstituto di Genetica e Biofisica ‘A. Buzzati Traverso’, CNR, and cDipartimento di Biologia Strutturale e Funzionale, Università di Napoli ‘Federico II’, Naples; dDipartimento di Scienze Pediatriche e dell’Adolescenza, Università di Torino, Torino, and eLaboratorio di Citogenetica e Genetica Molecolare, Istituto Auxologico Italiano, Milano, fDivision of Medical Genetics, San Paolo School of Medicine, University of Milan, Milan, Italy

Abstract The parent-of-origin-dependent expression of IGF2 and H19 is controlled by the imprinting center 1 (IC1) consisting of a methylation-sensitive chromatin insulator. IC1 is normally methylated on the paternal chromosome and nonmethylated on the maternal chromosome. We found that 22 cases in a large cohort of patients affected by Beckwith-Wiedemann syndrome (BWS) had IC1 methylated on both parental chromosomes, resulting in biallelic activation of IGF2 and biallelic silencing of H19. These individuals had marked macrosomia and high incidence of Wilms’ tumor. A subset of these patients had 1.4- to 1.8-kb deletions with hypermethylation of the remaining IC1 region and fully penetrant BWS phenotype when transmitted maternally. Another subset of individuals with IC1 hypermethylation had a similar clinical phenotype but no mutation in the local vicinity. All these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal epigenotype. Similarly, no IC1 deletion was detected in 10 nonsyndromic Wilms’ tumors with IC1 hypermethylation. In conclusion, methylation defects at the IGF2-H19 locus can result from inherited mutations of the imprinting center and have high recurrence risk or arise independently from the sequence context and not transmitted to the Copyright © 2009 S. Karger AG, Basel progeny.

There is increasing evidence that aberrant chromatin states leading to aberrant gene expression patterns (epimutations) have important roles in human disease [1]. However, the causes, heritability and relationship with phenotype of many of these

lesions are still undefined. For instance, although it is generally accepted that epigenetic marks are cleared between generations, there is a number of cases in which this seems not to be the case. The human disorders caused by defects of genomic imprinting provide a paradigm for studying these issues.

Genomic Imprinting

Genomic imprinting is an epigenetic mechanism causing the expression of a minority of genes to be monoallelic and dependent on their gametic origin [2]. Correct imprinting is required for normal development, while defective imprinting is associated with human disease [3]. A 1-Mb cluster of imprinted genes is present at chromosome 11p15.5 (fig. 1). The cluster is functionally divided into two domains that are autonomously controlled by separate imprinting control regions or imprinting centers (IC1 and IC2 [4, 5]). These are CpG-rich regions that work by different mechanisms, but share as a common feature to be differentially methylated on the maternally and paternally derived chromosomes (differentially methylated regions, DMRs). Two genes, insulin-like growth factor 2 (IGF2) and H19, are located in domain 1 of the 11p15.5 imprinted gene cluster. IGF2 is a paternally expressed fetal growth factor gene with an important role in cancer development [6]. H19 is a maternally expressed noncoding RNA with possible tumor-suppressor functions [7]. The reciprocal imprinting of IGF2 and H19 is controlled by IC1 in the majority of tissues. The function of this control element has been extensively studied in the mouse. IC1 (also known as H19 DMR) is a methylation-sensitive chromatin insulator located between IGF2 and H19 [8]. Its nonmethylated maternal allele interacts with the multi-zinc finger protein CTCF. This binding is required on the maternal chromosome for maintaining the nonmethylated status of the region and preventing the activation of the IGF2 promoter by downstream enhancers that activate the H19 gene instead. On the paternal chromosome, conversely, DNA methylation prevents CTCF binding at IC1 and allows the enhancer-mediated activation of IGF2 while the H19 promoter is hypermethylated and silenced. Recent evidences indicate that the methylationsensitive binding of CTCF at IC1 mediates higher-order chromatin conformations in a parent of origin-specific manner [9]. In particular, the two parental IC1 alleles interact with two different DMRs of Igf2 on the maternal and paternal chromosomes [10]. This may partition the maternal and paternal Igf2 alleles into inactive and active chromatin domains, respectively.

Beckwith-Wiedemann Syndrome

The Beckwith-Wiedemann syndrome (BWS, MIM 130650) is a developmental disorder characterized by variable clinical features, including overgrowth, macroglossia,

2

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

d H19

d INS d IGF2

d TH

d ASCL2

LTRPC5 TSSC4 CD81 TSSC6 d D D D

D KCNQ1

d KCNQ1OT1/LIT1

CARS NAP1L4 TSSC3 TSSC5 CDKN1C d d d D d

Domain 2

IC1

tel IC2

cen

Domain 1

Expressed from the paternal chromosome Expressed from the maternal chromosome Biallelic expression Imprinted expression not well defined DMR methylated on the paternal chromosome DMR methylated on the maternal chromosome

Fig. 1. The 11p15.5 imprinted gene cluster.

abdominal wall defects and increased incidence of embryonal tumors that is caused by defective expression of imprinted genes located on chromosome 11p15.5 [11]. The majority of the BWS cases are sporadic. The rare familial cases show a predominantly autosomal-dominant inheritance and preferential expression following maternal transmission. Heterogeneous molecular defects are found in BWS. Only 5% of the cases (40% of the familial ones) have typical single-gene defects, consisting in lossof-function mutations of CDKN1C. About 20% of the cases have uniparental paternal disomy (UPD) of 11p15.5 loci, indicating that BWS is caused by excess of imprinted genes expressed from the paternal chromosome and/or defect of imprinted genes expressed from the maternal chromosome. The majority of the other cases show DNA methylation defects at either IC1 or IC2. Similarly to BWS, patients with nonsyndromic Wilms’ tumor also have IC1 hypermethylation, but this is restricted to cancer tissues in these cases [12]. In this chapter, we review the clinical characteristics and molecular features of the cases of BWS and Wilms’ tumor with hypermethylation at IC1.

IC1 Microdeletions

Gain of methylation at IC1 is usually found in only 5–10% of the BWS cases [11, 13]. However, the high incidence of Wilms’ tumor-associated with these molecular abnormalities makes them particularly important to study. We found 1.4- to

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

3

1.8-kb deletions of the IC1 region in 9 BWS patients with IC1 hypermethylation [14–16]. These cases were part of dominant inheritance pedigrees with maternal transmission and characterized by high recurrence risk. The deletions remove 1–2 target sequences for CTCF (CTS) resulting in hypermethylation of the residual CTSs and cosegregate with the BWS phenotype with full penetrance if maternally inherited. In 2005, Prawitt et al. [17] described a further BWS family with a 2.2-kb deletion eliminating 3 CTSs from IC1. However, maternal transmission of this deletion was not necessarily associated with the BWS phenotype. Indeed, an additional mutation consisting in duplication of the 11p15 region was present in the affected children. Interestingly, this deletion did not alter the methylation of the flanking sequences, suggesting that the hypermethylation of the imprinting center significantly contribute to the BWS pathogenesis. We also observed that the mutant alleles with gain of methylation had abnormally spaced CTSs and proposed that the 1.4- to 1.8-kb deletions resulted in lowering the affinity of IC1 for CTCF [15].

IC1 Hypermethylation without Microdeletion

Neither deletion nor any other point mutation of the IC1 region was demonstrated in 13 patients of our cohort with IC1 hypermethylation [18, and data not shown]. These were all sporadic cases and in 2 of them, the maternal IC1 allele of the index patient segregated in 1 of his healthy relatives. A detailed methylation analysis showed that the hypermethylation was extended over the entire or only 3⬘ half of the IC1 region, did not affect other imprinted loci, generally occurred in the mosaic form and was never present in the unaffected relatives. The chromosome carrying the imprinting abnormality derived from either the maternal grandfather or maternal grandmother. These results indicate that, in the absence of deletions, IC1 hypermethylation generally occurs as sporadic epimutation and is associated with low recurrence risk.

Clinical Phenotype

The clinical features of the patients carrying IC1 deletions and those with IC1 hypermethylation without accompanying deletion are very similar. However, they differ significantly from the phenotypes of the BWS patients with other molecular defects [18]. Table 1 is a summary of the characteristics of a cohort of 132 individuals with clinical diagnosis of BWS [19], subdivided into 4 classes according to the molecular defect found [20]. Features, such as pronounced macrosomia, mild or absent defects of the abdominal wall and elevated incidence of Wilms’ tumor are evident among the individuals with IC1 hypermethylation.

4

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

Table 1. Clinical features of 132 BWS patients investigated for methylation defects at chromosome 11p15.5 Clinical features

IC1 hypermethylation IC2 11p15.5 Pat with and without hypomethylation UPD (n = 22) microdeletion (n = 43) (n = 20)

Normal methylation (n = 47)

Macrosomia (weight >90th centile) Polydramnios Macroglossia Rectum diastasis Umbilical hernia Exomphalos Inguinal hernia Hemihypertrophy Facial Asymmetry Hepato-/spleno-megaly Neonatal Hypoglycemia Ear pits/creases Facial dysmorphisms Naevus flammeous Renal abnormalities Wilms’ tumor Hepatoblastoma Other tumors Ureteral abnormalities VSD/ASD/PDA shunt Convulsions Mental retardation

16 (80)

17 (39)

8 (36)

22 (47)

5 (25) 17 (85) 12 (60) 10 (50) 0 (0) 1 (5) 7 (35) 4 (20) 12 (60) 8 (40) 7 (35) 7 (35) 5 (25) 11 (55) 5 (25) 0 (0) 0 (0) 3 (15) 1 (5) 1 (5) 1 (5)

8 (19) 42 (98) 20 (46) 17 (39) 14 (33) 4 (9) 14 (33) 8 (19) 12 (28) 18 (42) 30 (70) 20 (46) 27 (63) 7 (16) 0 (0) 0 (0) 1*(2) 2 (4) 2 (4) 5 (12) 6 (14)

6 (27) 19 (86) 9 (41) 7 (32) 1 (4) 0 (0) 16 (73) 1 (4) 9 (41) 10 (45) 7 (32) 6 (27) 8 (36) 9 (41) 1 (4) 2 (9) 0 (0) 2 (9) 0 (0) 0 (0) 3 (14)

3 (6) 38 (81) 28 (60) 17 (36) 4 (8) 3 (6) 14 (30) 6 (13) 6 (13) 8 (17) 26 (55) 18 (38) 14 (30) 10 (21) 1 (2) 0 (0) 0 (0) 2 (4) 2 (4) 2 (4) 8 (17)

Numbers in parentheses denote percent values. Percentages that significantly differ in one molecular subgroup of patients from the others are indicated in bold. * Neuroblastoma.

Epigenetic Mosaicism

We observed that all IC1 CTSs are completely and exclusively methylated on the paternal chromosome in normal leukocyte DNA while incomplete hypermethylation of the maternal allele is present in the BWS patients with the 1.4- to 1.8kb IC1 deletions and the majority of patients without deletions suggesting that this imprinting defect is generally present in the mosaic form [16, 18]. We did not observe a clear relationship between the extent of hypermethylation at the IGF2-H19 locus and the severity of the BWS phenotype. However, it is possible that

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

5

the degree of methylation of leukocyte DNA is insufficient to predict the extent of mosaicism in the whole body and tissues, which is more important as the BWS phenotype should be examined. The high incidence of hemihyperthrophy (table 1) suggests that at least some of the clinical features of the patients with IC1 deletion are influenced by mosaicism. Mosaicism can also explain the high variability of the clinical phenotype that is characteristic of this disorder. In addition, diagnostic problems may be encountered with individuals who may not have abnormal methylation in their leukocyte DNA. To avoid these problems, it may be useful in the future to analyze DNAs derived from more than one tissue (e.g. blood leukocytes and buccal mucosa).

Loss of IGF2-H19 Imprinting

It has been shown in the mouse that the CTSs within the IC1 element are required for its insulator function [21]. Furthermore, CTCF binding controls the interaction between IC1 and the Igf2 DMRs that are required to partition the Igf2 and H19 genes into transcriptionally active and inactive chromatin loops [9, 10]. We observed a gain of methylation at the IGF2 DMR0 and DMR2 in the majority of the patients with IC1 hypermethylation (with or without microdeletions) [16, 22, and data not shown]. In addition, biallelic activation of IGF2 and biallelic silencing of H19 could be demonstrated in many of these patients. It is therefore likely that the epigenetic alterations resulting from the microdeletions or sporadic epimutations of IC1 lead the maternal 11p15.5 locus to acquire high-order chromatin structures that are typical of the paternal chromosome and associated with IGF2 activation and H19 silencing (fig. 2).

Origin of the Imprinting Defects

In principle, imprinting defects at ICs can derive from failure of erasure, establishment or maintenance of the imprint [2]. The incomplete hypermethylation of the CTSs found in the BWS patients with 1.4- to 1.8-kb deletions indicates mosaicism for the imprinting defect and suggests that the methylation is acquired postzygotically and results from insufficient protection from de novo methylation of the mutated maternal IC1. In the BWS patients without microdeletions the chromosome with abnormal IC1 methylation derived from either the maternal grandfather or maternal grandmother. Since the majority of these cases also showed mosaic hypermethylation, it is likely that also in these cases the methylation defect is acquired at a postzygotic stage. However, an incomplete imprint erasure resulting in an unstable methylation cannot be excluded. Knocking-down of CTCF in mouse oocytes and in cultured cells results in gain of methylation of IC1 [23]. We ruled out the presence of a mutation of CTCF gene in

6

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

IGF2

H19 Normal

IGF2

H19

BWS with IC1 microdeletions

IGF2

DMR0

H19

DMR2

IC1

BWS without IC1 microdeletions

Fig. 2. Loss of IGF2-H19 imprinting as consequence of sporadic and inherited epimutations. The diagram shows the expression of the maternal and paternal IGF2 and H19 alleles in normal individuals and BWS patients with maternally inherited IC1 microdeletions or sporadic IC1 hypermethylation. The methylated or nonmethylated status of the IGF2 DMR0, IGF2 DMR2, seven IC1 CTSs and H19 promoter is indicated by filled and open lollipops, respectively. The enhancers are indicated by ovals.

the patients without IC1 deletions. It cannot be excluded, however, that a mutation is present in other modifier genes. Considering the sporadic nature of these cases, the possibility that IC1 hypermethylation occurs as consequence of stochastic events or environmental influence should also be envisaged [24].

Absence of IC1 Microdeletions in Non-Syndromic Wilms’ Tumor

The 11p15.5 imprinted gene cluster is frequently affected in Wilms’ tumors [12]. Either maternal deletion/paternal duplication (LOH) or IC1 hypermethylation coupled to H19 silencing and IGF2 activation (LOI) can be found in a high proportion of tumors. In addition, the individuals who have somawide IC1 hypermethylation or 11p15.5 paternal UPD represent the molecular subgroup of BWS patients showing the highest risk of developing Wilms’ tumor [13]. Consistent with these observations, we found that 5/20 BWS patients with IC1 hypermethylation in our cohort had developed this neoplasm. Two of these had IC1 microdeletions [18]. However, no

Epimutations at the IGF2-H19 Locus in BWS and Wilms’ Tumor

7

IC1 deletion was found in 40 sporadic non-syndromic Wilms’ tumors. A previous American study demonstrated the absence of point mutations in the IC1 sequence of a series of sporadic Wilms’ tumors [25]. Overall, these data suggest that IC1 imprinting defects are generally not associated with a mutation in cis in non-syndromic Wilms’ tumor, as well as sporadic BWS. We recently demonstrated the presence of different methylation abnormalities at IGF2 in Wilms’ tumors and BWS, suggesting that sporadic imprinting defects arise through different mechanisms in neoplastic and non-neoplastic cells [22]

Conclusion

Although the common hallmark and probably the ultimate cause of the imprinting defects at the IGF2/H19 locus is represented by hypermethylation of IC1, our studies demonstrate that this epigenetic abnormality can result from more than one mechanism in BWS and Wilms’ tumor. In a first group of patients, we found that the epimutation is a direct consequence of a mutation in cis, consisting of a deletion of 1–2 CTSs. In these cases, the methylation defect and disease phenotype are reproduced whenever the mutation is transmitted through the maternal germline. In another group of patients, who carry no IC1 deletion, the epimutation is independent of the local DNA sequence and generally not transmitted to the progeny. Sporadic BWS and non-syndromic Wilms’ tumor belong to the second group of cases. Despite these differences, the IC1 epimutation is generally present in the patients in the mosaic form and probably acquired by postzygotic de novo methylation, providing an example of how intricate the relationship between genotype and epigenotype can be.

Acknowledgments This work was supported by grants from MIUR PRIN 2005, Istituto Superiore di Sanità, Associazione Italiana Ricerca sul Cancro, Telethon-Italia Grant No. GGP07086. F.C. was recipient of a fellowship from Società Italiana di Cancerologia and Fondazione Pezcoller.

References 1 2

3

8

Feinberg AP: Phenotypic plasticity and the epigenetics of human disease. Nature 2007;447:433–440. Reik W, Walter J: Genomic imprinting: parental influence on the genome. Nat Rev Genet 2001;2:21– 32. Arnaud P, Feil R: Epigenetic deregulation of genomic imprinting in human disorders and following assisted reproduction. Birth Defects Res [C] 2005; 75:81–97.

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Ainscough JFX, Koide T, Tada M, Barton S, Surani MA: Imprinting of Igf2 and H19 from a 130 kb YAC transgene. Development 1997;124:3621–3632. Cerrato F, Sparago A, Di Matteo I, Zou X, Dean W, Sasaki H, Smith P, Genesio R, Bruggemann M, Reik W, Riccio A: The two-domain hypothesis in BeckwithWiedemann syndrome: autonomous imprinting of the telomeric domain of the distal chromosome 7 cluster. Hum Mol Genet 2005;14: 503–511.

Riccio · Sparago · Verde · De Crescenzo · Citro · Cubellis · Ferrero · Silengo · Russo · Larizza · Cerrato

6 LeRoith D, Roberts, CTJr: The insulin-like growth factor system and cancer. Cancer Lett 2003;195:127– 137. 7 Hao Y, Crenshaw T, Moulton T, Newcomb E, Tycko B: Tumour-suppressor activity of H19 RNA. Nature 1993;365:764–767. 8 Hark AT, Schoenherr CJ, Katz DJ, Ingram RS, Levorse JM, Tilghman SM: CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 2000;405:486–489. 9 Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao Z, Lobanenkov V, Reik, W, Ohlsson R: CTCF binding at the H19 imprinting control region mediates maternally inherited higherorder chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA 2006; 103:10684–10689. 10 Murrell A, Heeson S, Reik W: Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops. Nat Genet 2004;36:889–893. 11 Weksberg R, Shuman C, Smith AC: BeckwithWiedemann syndrome. Am J Med Genet [C] 2005; 137:12–23. 12 Feinberg AP, Cui H, Ohlsson R: DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Semin Cancer Biol 2002; 12:389–398. 13 Cooper WN, Luharia A, Evans GA, Raza H, Haire AC, Grundy R, Bowdin SC, Riccio A, Sebastio G, Bliek J, Schofield PN, Reik W, Macdonald F, Maher ER: Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome. Eur J Hum Genet 2005;13:1025–1032. 14 Sparago A, Cerrato F, Vernucci M, Ferrero GB, Cirillo Silengo M, Riccio A: Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann. Nat Genet 2004;36:958– 960. 15 Cerrato F, Sparago A, Farina L, Ferrero GB, Cirillo Silengo M, Riccio A: Reply to Microdeletion and IGF2 loss of imprinting in a cascade causing Beckwith-Wiedemann syndrome with Wilm’s tumor. Nat Genet 2005;37:786–787. 16 Sparago A, Russo S, Cerrato F, Ferraiuolo S, Castorina P, Selicorni A, Schwienbacher C, Negrini M, Ferrero GB, Silengo MC, Anichini C, Larizza L, Riccio A: Mechanisms causing imprinting defects in familial Beckwith-Wiedemann syndrome with Wilms’ tumour. Hum Mol Genet 2007;16; 254–264.

17 Prawitt D, Enklaar T, Gartner-Rupprecht B, Spangenberg C, Oswald M, Lausch E, Schmidtke P, Reutzel D, Fees S, Lucito R, Korzon M, Brozek I, Limon J, Housman DE, Pelletier J, Zabel B: Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms’ tumor. Proc Natl Acad Sci USA 2005;102:4085–4090. 18 Cerrato F, Sparago A, Verde G, De Crescenzo A, Citro V, Cubellis MV, Rinaldi MM, Boccuto L, Neri G, Magnani C, D’Angelo P, Collini P, Perotti D, Sebastio G, Maher ER, Riccio A: Different mechanisms cause imprinting defects at the IGF2/H19 locus in BeckwithWiedemann syndrome and Wilms’ tumour. Hum Mol Genet 2008;17:1427–1435. 19 DeBaun MR, Tucker MA: Risk of cancer during the first four years of life in children from The BeckwithWiedemann Syndrome Registry. J Pediatr 1998;132: 398–400. 20 Priolo M, Sparago A, Mammì C, Cerrato F, Laganà C, Riccio A: MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a single-tube experiment. Eur J Hum Genet 2008;16:565–571. 21 Schoenherr CJ, Levorse JM, Tilghman SM: CTCF maintains differential methylation at the Igf2/H19 locus. Nat Genet 2002;33:66–69. 22 Murrell A, Ito Y, Verde G, Huddleson J, Woodfinel K, Cirillo Silengo M, Spreafico F, Perotti D, De Crescenzo A, Sparago S, Cerrato F, Riccio A: Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer. PloS ONE 2008;3:e1849. 23 Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS: Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 2004;303:238–240. 24 Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003;33(suppl): 245–254. 25 Cui H, Niemitz EL, Ravenel JD, Onyango P, Brandenburg SA, Lobanenkov VV , Feinberg AP: Loss of imprinting of insulin-like growth factor-II in Wilms’ tumour commonly involves altered methylation but not mutations of CTCF or its binding site. Cancer Res 2001;61:4947–4950.

Andrea Riccio Dipartimento di Scienze Ambientali Seconda Università di Napoli, Via Vivaldi, 43 IT–81100 Caserta (Italy) Tel. +39 0823 274 599, Fax +39 082 274 605, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 10–19

Epigenetic Regulation of Growth: Lessons from Silver-Russell Syndrome Thomas Eggermann Institute of Human Genetics, University Hospital, RWTH Aachen, Germany

Abstract Human growth is a complex process and requires the appropriate interaction of many members. Central members in the growth axes are regulated epigenetically and thereby reflect the profound significance of imprinting for correct mammalian ontogenesis. A prominent imprinting disorder, Silver-Russell syndrome (SRS), is a congenital disease characterized by intrauterine and postnatal growth retardation and other features. SRS represents the first human disorder with imprinting disturbances affecting two different chromosomes: nearly one tenth of patients carry a maternal uniparental disomy of chromosome 7 and more than 38% show a hypomethylation in the imprinting control region 1 in 11p15. Interestingly, hypermethylation of the same region is associated with the overgrowth disease Beckwith-Wiedemann syndrome (BWS), thus SRS and BWS can be regarded as genetically (and clinically) opposite diseases. Because of the different imprinting regions involved, SRS is a suitable model to decipher the role of imprinting in growth and the functional interaction Copyright © 2009 S. Karger AG, Basel between imprinted genes in different genomic regions.

Growth is a complex process with both genetics and environment contributing in equally parts. Among the involved genes is a substantial number of so-called imprinted factors, e.g. these genes are expressed only from one chromosome from one parent. Generally, paternally expressed imprinted genes enhance fetal growth, whereas maternally expressed ones suppress it. Based on this observation, the genetic conflict theory has been hypothesized: it explains the evolution of imprinted genes by paternally derived genes that aim at extracting more resources from the mother whereas maternally derived genes have to balance the nutrient provision to the current fetus with that to future fetuses of the same mother [1]. With the identification of human diseases caused by epigenetic mutations, the significance of a balanced expression of imprinted genes became obvious. In addition to the profound role of imprinted genes in congenital disorders, there is evidence for epigenetic inheritance in humans which might explain the transgenerational effects of grandparental nutrition on health in next generations [2].

The close relationship between imprinting and (fetal) growth is illustrated by the recent identification of specific genetic and epigenetic alterations in Silver-Russell syndrome (SRS)(OMIM 180860). SRS is a congenital disorder mainly characterized by pre- and postnatal growth restriction. The children are relatively macrocephalic and their face is triangular-shaped with a broad forehead and a pointed, small chin. In many cases, asymmetry of limbs and body and clinodactyly of the fifth digit is present. Growth failure is often accompanied by severe failure to thrive, and feeding difficulties are frequently reported. The latter is the main clinical concern and much effort is expended in encouraging adequate feeding. For those children without catch-up growth by the age of 2, growth hormone therapy is encouraged.

Genetics of SRS

The observation of ‘classical’ genetic findings in SRS patients such as families with several affected members and cytogenetic aberrations indicated the influence of genetic factors in the etiology of SRS. Among the rare SRS families, autosomalrecessive as well as dominant inheritance have been reported. This genetic heterogeneity has also been confirmed by the observation of different chromosomal disturbances (fig. 1). Whereas in other genetic diseases these findings help to identify the specific genes, in SRS the majority of the putative genetic observations did not enlighten the molecular basis of the disorder but indeed led to more confusion. This confusion was increased by the finding that the majority of monozygotic twins are discordant (for review, see ref. [3]). In circumstances in which genetic information is incomplete, twin studies are often helpful in establishing genetic causes. In SRS, these discordant monozygotic twins were misleading for a long time but can now be explained by the data from Gicquel et al. [4] who reported on discordant monozygotic twins carrying the same 11p15 epimutation but with mosaic distribution in different tissues. Despite the heterogeneous cytogenetic findings, aberrations of four chromosomal regions have been described for several times and these regions were therefore considered to harbor SRS relevant genes.

Chromosomes 15 and 17

The identification of deletions in 15q26qter in patients with SRS features led to the assumption that an imbalanced expression of the insulin-like growth factor 1 receptor gene (IGF1R) might cause SRS. Nevertheless, pathogenic mutations in IGF1R were not found in SRS patients [5]. Based on balanced translocations in 2 patients involving 17q24-q25, a central role of this chromosomal region in SRS etiology had been long discussed. However,

(Epi)Genetics of Silver-Russell Syndrome

11

3 1

5

4

2 …

6

7

…

…

13

14

19

20

Deletion

15

…

8

9

…

11

10

16

17

21

22

UPD

12

X

18

Y

Isochromosome/marker chromosome

Duplication/trisomy Translocation breakpoint

Ring chromosome

… more than two cases

Fig. 1. Chromosomal aberrations in SRS: Review on types, frequencies and affected genomic regions. A complete list of the literature describing chromosomal aberrations in SRS is available on request.

characterization of the 17q breakpoints in both patients showed that they were not identical [6]. Furthermore, the previously reported heterozygous deletions in the growth hormone (GH) gene cluster in 17q are now regarded as apathogenic polymorphisms [7].

Chromosome 7

Cytogenetic aberrations of chromosome 7 including duplications of 7p11.2p13 and small marker chromosomes have been identified in several SRS individuals (for review, see ref. [8]) and lead to the assumption that a ‘SRS gene’ should be localized in

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the short arm of chromosome 7 (see below). However, the most spectacular finding was that of maternal uniparental disomy of chromosome 7 (UPD(7)mat) in 7–10% of SRS individuals [9]. Uniparental disomy (UPD) is the inheritance of two homologous chromosomes or chromosomal fragments (partial UPD) from only one parent in an euploid offspring. Whole chromosome UPD can be the result of meiotic as well as mitotic non-disjunction errors, but the most frequent mode of formation is via trisomic rescue: 10–15% of all recognized human conceptions are trisomic and most of them are lethal. For many chromosomes, the only way that the trisomic conception survives is that one of the three chromosomes is lost. In one third of these cases a random chromosome loss might lead to UPD. If the UPD results from a postzygotic mitotic error, it might be associated with chromosomal mosaicism. Identification of UPD is extremely helpful because it helps deciphering imprinted regions and genes. UPD has meanwhile been described for the majority of chromosomes but only chromosomes 6, 7, 11, 14 and 15 show a specific phenotype on the basis of imprinting disturbances. So far, research on chromosome 7 encoded factors has focused on two chromosomal segments. For the candidate region in 7p11.2-p13 SRS patients with duplications have been reported (for review, see ref. [10]). The region harbors an imprinted gene (growth factor receptor bound protein 10/GRB10, see below) and several factors involved in human growth and development (fig. 2). Pathogenic mutations in these genes have not yet been described in SRS (for review, see ref. [8]). The most prominent candidate in 7p is GRB10 which encodes a cytoplasmic adaptor protein and interacts with tyrosine kinase receptors. The gene shows a complex expression in mammalian tissues with various isoforms expressed either from the maternal or from the paternal copy. In mice, Grb10 is mainly expressed from the maternal allele; the loss of maternal Grb10 results in both fetal and placental overgrowth [11, 12] demonstrating its role as a growth suppressor. By contrast, Grb10 overexpression in mice causes postnatal growth retardation and insulin resistance [13]. Grb10 serves a negative regulator of insulin signaling and action in vivo [12, 13] but a role in a fetal growth pathway independent of Igf2 has also been postulated [11]. To summarize, Grb10/GRB10 plays an essential role in growth and is therefore still a good candidate for SRS. Nevertheless, neither point mutations in the coding region nor aberrant methylation of GRB10 have been detected in SRS patients despite extensive screening studies (for review, see ref. [8]). There is additional evidence that the chromosomal region 7q31 is also involved in the etiology of SRS: meanwhile four growth-restricted patients have been described with segmental maternal UPD of the long arm of chromosome 7, three of which with SRS features (fig. 2). In 7q31, three imprinted genes (MEST/PEG1; CPA4; COPG2) and two imprinted noncoding RNAs (MESTIT1, CIT1/COPG2IT1) are localized but screening studies did not detect any pathogenic variants.

(Epi)Genetics of Silver-Russell Syndrome

13

22

Candidate genes

21

p

15 14 13

Fig. 2. SRS candidate genes on chromosomes 7 and review on the extents of segmental UPD(7)mat and maternal duplications in 7p12 reported so far (* imprinted genes). IGFBP1, IGFBP3 insulin-like growth factor-binding proteins 1 and 3; PHKG1 = Phosphorylase kinase 1; EGFR = epidermal growth factor receptor; GHRHR = growth hormone-releasing hormone receptor; MEST/PEG1 = mesoderm-specific transcript; CPA4 = carboxypeptidase A4; COPG2 = coatomer protein complex subunit gamma 2; MESTIT MEST = intronic transcript; CIT1/COPG2IT1 COPG2 = intronic transcript; PAX4 = paired box gene 4.

12 11.2 11.1 11.1

Duplications Segmental UPD(7)mat

GHRHR IGFBP1 IGFBP3 GRB10*

EGFR PHKG1 c7orf10

11.2

21

q

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31

32 33 34 35 36

MEST* MESTIT1*

CIT1* COPG2*

PAX4 CPA4

Chromosome 7

Chromosome 11

Recent exciting findings indicate an important role of the region 11p15 in the etiology of SRS (fig. 3). The first hint was the identification of maternal 11p15 duplications in growth retarded patients and more or less striking SRS features (for review: [14]). Interestingly, the opposite disturbance – duplication of paternal 11p15 – is associated with the overgrowth disease Beckwith-Wiedemann syndrome (BWS, OMIM 130650). Numerous genetic and epigenetic alterations can be detected in BWS patients (table 1; fig. 3) (for review, see ref. [15]) but in more than 50% aberrant methylation patterns in 11p15 are involved. The search for epimutations in 11p15 in SRS patients was therefore consequent and indeed, hypomethylation at the telomeric imprinting control region 1 (ICR1) in 11p15 regulating H19 and IGF2 expression could be identified in more than 38% of cases (table 1; fig. 3). A comparison of the 11p15 disturbances in BWS and SRS reveals many similarities in the molecular biology of the two disorders.

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Table 1. 11p15 (epi)mutations in SRS and BWS and their frequencies: modified from Eggermann et al. [8] Type of mutation

SRS

Frequencies

BWS

Frequencies

In ICR1

hypomethylation

>38%

hypermethylation

2–8%

In ICR2

mutations in CKDN1C

–

mutations in CDKN1C

5–10% sporadic 40% autosomal dominant traits

hypermethylation

–

hypomethylation

50–60%

maternal duplication

1 case

11p structural rearrangements

maternal duplications

4 cases

paternal duplications

1–2%

UPD of 11p15

maternal

?

paternal UPD

10–20%

Others

maternal UPD7/7p 10% duplications >40% unknown

unknown

10–20%

First of all, epimutations affect the ICR1 in both entities. In BWS, hypermethylation of the ICR1 leads to biallelic expression of IGF2, potentially doubling the IGF2 dose in specific organs. By contrast, the ICR1 hypomethylation observed in SRS should result in a suppressed IGF2 expression in the target tissues. As a result of its central role in human growth (and tumorigenesis), IGF2 is the perfect candidate for both diseases, and indeed IGF2 is highly expressed in tissues which are affected in BWS. In vitro, IGF2 expression is altered in both syndromes: in tissue samples from BWS patients IGF2 is overexpressed at the mRNA level (for review, see ref. [15]), in fibroblasts of SRS individuals IGF2 mRNA is reduced [4]. However, IGF2 serum levels in both BWS patients and SRS patients with H19 hypomethylation are normal [16, 17] but we have to consider that the liver as the major organ of postnatal IGF2 secretion expresses the factor from a nonimprinted promoter. Due to the negative findings in serum it has been concluded that ICR1 demethylation does not directly influence IGF2 secretion in SRS children but an altered IGF2 production probably leads to a diminished autoparacrine action in the fetus. The recent identification of a SRS patient with a duplication restricted to the centromeric imprinting control region 2 (ICR2) [18] which regulates the expression of CDKN1C, KCNQ1 and further genes suggests that both ICRs on 11p15 are involved in the aetiology of the disease, like in BWS where epi/mutations in the ICR2 account for more than 50% of cases (table 1). This finding and further data obtained from BWS patients and mice models suggest that ICR1 and ICR2 interact (for review, see ref. [8]).

(Epi)Genetics of Silver-Russell Syndrome

15

11pter

11cen CDKN1C

KCNQ1

IGF2

M

H19

CTCF

KCNQ1OT1 CH3

CH3

CH3 CH3

CDKN1C

KCNQ1

IGF2

H19

P KCNQ1OT1

BWS

ICR2

SRS

ICR1

Chromosomal rearrangements (duplications) CDKN1C

M

KCNQ1

IGF2

H19

IGF2

H19

IGF2

H19

M

KCNQ1OT1

CDKN1C

P

KCNQ1

M

KCNQ1OT1

CDKN1C

P

KCNQ1

P

KCNQ1OT1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

IGF2

H19

IGF2

H19

IGF2

H19

KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

Uniparental disomy (UPD) CDKN1C

KCNQ1

P

IGF2

H19

IGF2

H19

KCNQ1OT1

CDKN1C

P

KCNQ1 KCNQ1OT1

Altered Methylation of ICR1

M P

CDKN1C

KCNQ1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

M

CDKN1C

P

KCNQ1OT1

KCNQ1 KCNQ1OT1

KCNQ1OT1

CDKN1C

KCNQ1 KCNQ1OT1

Duplication of ICR2

(Epi)mutations of/in ICR2 Hypomethylation of ICR2 M

CDKN1C

P

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

KCNQ1OT1

KCNQ1

M

CDKN1C

KCNQ1

CDKN1C

KCNQ1

KCNQ1OT1

P

CDKN1C

M P

CDKN1C

KCNQ1

CDKN1C

KCNQ1

IGF2

H19

IGF2

H19

H19

KCNQ1

IGF2

H19

KCNQ1OT1

KCNQ1OT1

Pointmutations in CDKN1C

IGF2

KCNQ1OT1

Expressed

Silenced

IGF2

IGF2

CDKN1C

CDKN1C

KCNQ1OT1 KCNQ1OT1

Fig. 3. Epigenetic regulation of the two imprinting center regions (ICR) in 11p15 and the different (epi)mutations affecting 11p15 in BWS and SRS. CDKN1C = Cyclin-dependent kinase inhibitor/p57KIP2; KCNQ1 = potassium channel KQT-family member 1; KCNQ1OT1 KCNQ1 = intronic transcript 1; IGF2 = insulin-like growth factor 2.

Do Different Mutations in Functional Networks Explain the Heterogeneity of SRS?

When looking at the major chromosomal regions involved in rearrangements in SRS we see that three of them, i.e. chromosomes 7, 11 and 15, harbor members of the IGF system, a mediator of pre- and postnatal growth. SRS might therefore represent a common phenotype caused by alterations in members of this axis such as the ligand IGF2, its receptor IGF1R and the IGF signaling modulator GRB10. The effects of IGF2 on fetal growth are mainly mediated through the IGF1R. Mice knockout models indicate that loss of the paternal IGF2 causes IUGR with reduction in 40% of the body weight in newborn mice [19] whereas experimental overexpression of the same allele results in hypertrophic mice [20].

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Another axis – an ‘imprinted gene network’ – has recently been hypothesized by Arima et al. [21]. They observed that in vitro the imprinted gene PLAGL1/ZAC (pleomorphic adenoma gene-like 1) in 6q24 drives the expression of KCNQ1OT1 noncoding RNA in 11p15. PLAGL1/ZAC might affect imprinted expression along the ICR2 domain in 11p15, including CDKN1C. This would put PLAGL1/ZAC, KCNQ1OT1 and CDKN1C in a common regulatory mechanism that influences intrauterine growth. According to Arima et al. [21], the assumed regulation of CDKN1C by PLAGL1/ZAC indicates a potential role of this gene in BWS but they did not detect aberrant methylation at the PLAGL1/ZAC DMR in a small number of BWS patients. Indeed they observed changes in the methylation of the ICR2 in two patients with transient neonatal diabetes mellitus (TNDM) and 6q24 disturbances.

Genotype-Phenotype Correlation in SRS?

With the identification of the ICR1 hypomethylation in 11p15 and UPD(7)mat the molecular confirmation of the clinical diagnosis of SRS is now possible in ~50% of patients (table 1). However, we have to bear in mind that routine diagnostics is based on lymphocytes and that nearly all SRS patients with ICR1 hypomethylation are mosaics. Thus, we hypothesize that a subgroup of patients escapes molecular diagnosis because their mosaicism affects other tissues than blood cells. In these cases, the mosaic distribution of the epimutation probably influences the phenotype. This assumption is supported by the findings of several groups [4, 22, 23] who reported on a correlation between the degree of methylation at the ICR1 locus and the severity of the phenotype. The finding of different epigenetic and genetic alterations in SRS patients should enable us to delineate a genotype-phenotype correlation. Indeed, the SRS phenotype of carriers of the 11p15 epimutation is generally more severe and typical than that of UPD(7)mat carriers or those SRS without known mutation [16, 24] but exceptions exist [23]. Additionally, in single cases chromosomal anomalies might also influence the phenotype depending on the chromosomal regions involved. In total, the phenotypic transition is fluent and therefore carriers of 11p15 epimutations and UPD(7) mat can not be discriminated solely by clinical findings. We therefore suggest to test all patients with intrauterine and severe postnatal growth retardation and only slight signs reminiscent for SRS for the known mutations. Screening of larger groups of growth-retarded patients will help to establish the frequency of 11p15 epimutation and UPD(7)mat in this heterogeneous cohort. Based on the current data it can be hypothesized that the frequency of SRS is underestimated because of the difficult and often subjective clinical diagnosis which depends on the experience of the clinical investigator. As aforementioned, IGF2 serum levels are within the normal range in SRS patients possibly due to a biallelic expression in the liver whereas its predominantly paternal

(Epi)Genetics of Silver-Russell Syndrome

17

expression in the fetus makes functional consequences of an aberrant methylation possible. Recently, Binder et al. [24] demonstrated that IGF1 serum levels in 11p15 epimutation carriers are inadequately high in comparison to non-syndromic short children. Moreover, IGFB3 levels were increased in the 11p15 patients while UPD(7) mat patients showed regular IGF1 and IGFB3 serum levels. It was therefore concluded that heterogeneity of SRS does not only include the genetic and clinical level but also hormone regulation.

Conclusion

In the highly developed countries ~3% of children are born too small and 20% of them do not show catch-up growth, patients with SRS belonging to the latter group. The functional cause for the persisting growth retardation is currently unknown in the majority of cases, and the only pharmacological option available is treatment with hGH. Since the response is highly variable even in a defined cohort like SRS patients, a more specific classification of the patients based on genetic methods might help to understand this variable response and allow to specifically adapt the treatment. Thus, SRS can be regarded as a model to decipher the functional link between the different genetic and epigenetic factors identified in growth retarded individuals; future studies will further enlighten the complex interactions between growth factors and contribute to a better directed therapy.

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Moore T, Haig D: Genomic imprinting in mammalian development: a parent tug-of-war. Trends Genet 1991;7:45–49. Pembrey ME, Bygren LO, Kaati G, Edvinsson S, Northstone K, Sjöström M, Golding J, ALSPAC Study Team: Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet 2006; 14: 159–166. Bailey W, Popovich B, Jones KL: Monozygotic twins discordant for the Russell-Silver syndrome. Am J Med Genet 1995;58:101–105. Gicquel C, Rossignol S, Cabrol S, Houang M, Steunou V, Barbu V, Danton F, Thibaud N, Le Merrer M, Burglen L, Bertrand AM, Netchine I, Le Bouc Y: Epimutation of the telomeric imprinting center region on chromosome 11p15 in SilverRussell syndrome. Nat Genet 2005;37:1003–1007. Binder G, Mavridou K, Wollmann HA, Eggermann T: Screening for insulin-like growth factor-I receptor mutations in patients with Silver-Russell syndrome. J Pediatr Endocrinol Metab 2002;15; 1167–1171.

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Dörr S, Midro AT, Farber C, Giannakudis J, Hansmann I: Construction of a detailed physical and transcript map of the candidate region for Russell- Silver syndrome on chromosome 17q23q24. Genomics 2001;71:174–181. Eggermann T, Schoenherr N, Eggermann K, Wollmann HA: Hypomethylation in the 11p15 telomeric imprinting domain in a Silver-Russell patient with a CSH1 deletion (17q24) renders a functional role of this alteration unlikely. J Med Genet 2007; 44:e77. Eggermann T, Eggermann K, Schönherr N: Growth retardation versus overgrowth: Silver-Russell syndrome is genetically opposite to BeckwithWiedemann syndrome. Trends Genet 2008;24: 195–204. Kotzot D, Schmitt S, Bernasconi F, et al: Uniparental disomy 7 in Silver-Russell syndrome and primordial growth retardation. Hum Mol Genet 1995;4:583– 587.

Eggermann

10 Monk D, Bentley L, Hitchins M, Myler RA, ClaytonSmith J, Ismail S, Price SM, Preece MA, Moore GE: Chromosome 7p disruptions in Silver-Russell syndrome: delineating an imprinted candidate gene region. Hum Genet 2002;111:376–387. 11 Charalambous M, Smith FM, Bennett WR, Crewe TE, MacKenzie F, Ward A: Disruption of the imprinted Grb10 gene leads to disproportionate overgrowth by an Igf2-independent mechanism. PNAS 2003;100:8292–8297. 12 Wang L, Balas B, Christ-Roberts CY, Kim RY, Ramos FJ, Kikov CK, Li C, Deng C: Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo. Mol Cell Biol 2007;27:6497– 6505. 13 Shiura H, Reyna S, Muri N, Dony LQ, De Fronzo RA, Liu F: Meg1/Grb10 overexpression causes postnatal growth retardation and insulin resistance via negative modulation of the IGF1R and IR cascades. BBRC 2005;329; 909–916. 14 Eggermann T, Meyer E, Obermann C, Heil I, Schuler H, Ranke MB, Eggermann K, Wollmann HA: Is maternal duplication 11p15 associated with Silver-Russell syndrome? J Med Genet 2005;42:e26. 15 Weksberg K, Smith AC, Squire J, Sadowski P: Beckwith-Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum Mol Genet 2003;12:R61–R68. 16 Netchine I, Rossignol S, Dufourg MN, Azzi S, Rousseau A, Perin L, Houang M, Seunou V, Esteva B, Thibaud N, Demay M-CR, Danton F, Petricko E, Bertrand A-M, Heirnichs C, Carel J-C, Loeuille G-A, Pinto G, Jaquemont M-L, Gicquel C, Cabrol S, Le Bouc Y: 11p15 ICR1 loss of methylation is a common and specific cause of typical Russell-Silver syndrome: clinical scoring system and epigeneticphenotypic correlations. J Clin Endocrinol Metabol 2007;92: 3148–4154.

17 Schneid H, Vazquez MP, Vachev P, Gournelen M, Cabrol S, Le Bouc Y: The Beckwith-Wiedemann syndrome phenotype and the risk of cancer. Med Pediatr Oncol 1997;28; 411–415. 18 Schönherr N, Meyer E, Schmidt A, Wollmann HA, Eggermann T: The centromeric 11p15 imprinting center is also involved in Silver-Russell syndrome. J Med Genet 2007;44:59–63. 19 DeChiara TM, Efstratiadis A, Robertson EJ: A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78–80. 20 Sun F-L, Dean WC, Kelsey G, Allen ND, Reik W: Transactivation of Igf2 in a mouse model of Beckwith-Wiedemann syndrome. Nature 1997;389: 809–815. 21 Arima T, Kamikara T, Hayashita T, Kato K, Inoue T, Shirayashi Y, Oskimura M, Soejima H, Makai T, Wake N: ZAC, Lit1 (KCNQ1OT1) and p57KIP2 (CKDN1C) are in an imprinted gene network that might play a role in Beckwith-Wiedemann syndrome. Nucleic Acids Res 2005;33:2650–2660. 22 Bliek J, Terhal P, van den Bogaard M-J, Maas S, Hamel B, Salieb-Beugelaar G, Simon M, Letteboer T, van der Smagt J, Kroes H, Mannens M: Hypomethylation of the H19 gene causes not only Silver-Russell syndrome (SRS) but also isolated asymmetry or an SRS-like phenotype. Am J Hum Genet 2006;78:604–614. 23 Zeschnigk M, Albrecht B, Buiting K, Kanber D, Eggermann T, Binder G, Gromoll J, Prott E-C, Seland S, Horsthemke B: IGF2/H19 hypomethylation in Silver-Russell syndrome and isolated hemihypoplasia. Eur J Hum Genet 2008;16:328–334. 24 Binder G, Seidel A-K, Martin DD, Schweizer R, Schwarze P, Wollmann HA, Eggermann T, Ranke MB: The endocrine phenotype in Silver-Russell syndrome is defined by the underlying epigenetic alteration. J Clin Endocrinol Metab 2008;93:1402– 1407.

Thomas Eggermann, PhD Institute of Human Genetics University Hospital, RWTH Aachen Pauwelsstrasse 30, DE–52074 Aachen (Germany) Tel. +49 241 808 8008, Fax +49 241 808 2394, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 20–28

Genetic Imprinting: The Paradigm of Prader-Willi and Angelman Syndromes Fiorella Gurrieri ⭈ Maria Accadia Institute of Medical Genetics, Catholic University, Rome, Italy

Abstract Imprinted genes are expressed from only one of the two parental alleles. A consequence of genomic imprinting is that viable embryos must receive two haploid genome complements from parents of opposite sex. The parental-specific expression is obtained through epigenetic modifications (DNA methylation, histone tail modifications) which alter the conformation of chromatin fiber and therefore regulate the expression of the underlying genes. Deletions, duplication, mutations or alterations of imprinting of the only active allele, as well as uniparental disomy or loss of imprinting of the inactive allele lead to an unbalance (loss of function or gain of function) in the dosage of the gene product and may have phenotypic consequences. Two such examples in human pathology are represented by the Prader-Willi and Angelman syndromes, whose phenotypes result from loss of paternal or maternal contribution of the 15q11-q13 genomic region, respectively. Prader-Willi syndrome is characterized by pre- and postnatal hypotonia, feeding difficulties in early life and subsequent hyperphagia with obsessive/compulsive food searching, obesity, short stature, hypogonadism and acromicria. Psychomotor development is mildly affected and behavioral problems are more relevant. Patients with Angelman syndrome show a completely different phenotype characterized by severe mental retardation, absent speech, autistic-like behavior, severe epilepsy and postnatal Copyright © 2009 S. Karger AG, Basel microcephaly.

The term ‘genomic imprinting’ refers to the different expression of an allele depending on its parent-specific origin, so that only the paternal or the maternal allele is expressed in certain tissues or developmental stages. A direct consequence of this process is a physiologic functional haploidy of imprinted genes, which makes them more likely to be associated with disease. Normal development in mammals requires genes to be inherited from both parents. The functional differences between the maternal and paternal genomes are clearly recognized in two human tumors: the ovarian teratoma, arising from ovarian germ cells, and the hydatidiform mole, arising from cytotrophoblast. The ovarian teratoma has a gynogenetic origin and contains elements derived from all three germinal layers (ectoderm, mesoderm, endoderm)

[1], whereas the hydatidiform mole has an androgenetic origin and is composed by extra-embryonic trophoblast elements [2]. Nuclear transplantation experiments in mouse provided further evidence of the different developmental program of the maternal and paternal genome in embryo. Mouse eggs manipulated to contain two maternal pronuclei (gynogenotes) showed better formation of the embryo, but very poor development of extraembryonic tissues. In contrast, in mouse eggs with two paternal pronuclei (androgenotes), an exuberant trophoblast growth and failure of embryonic development was noted. These experiments pointed out that the maternal and paternal contributions to the embryonic genome in mammals were not equivalent and the correct development of the embryo required both of them [3]. It was also noted that this parental effect did not involve the whole genome, but was limited to specific chromosomal regions containing clusters of imprinted genes, which were differentially marked in the maternal and paternal germ lines [4].

Meaning of Epigenetic Reprogramming

Genomic imprinting is an epigenetic process. But what does ‘epigenetic’ mean? Actually, this term has been reinvented twice in the history of biology and development. The term epigenetic was first introduced in 1942 by Conrad Hal Waddington to describe biological differences between tissues that result from the process of development. He used the metaphor ‘genetic landscape’, suggesting that cell fates were established in development in a way similar to a marble rolling down to the lowest point of a ground in which ridges and furrows delineate the path and make it irreversible. Therefore, such a path depends not only on the intrinsic nature of the marble, but also on the conditioning through external forces [5]. Today, the term epigenetic refers to the modification of a given DNA trait, not changing its sequence, but rather its function through DNA methylation and histone tail modifications. Such modifications alter the conformation of the chromatin fiber, interfering with the transcriptional machinery and with DNA-binding proteins thus regulating the expression of the underlying genes [6]. This process is very important during the development of a multicellular organism, in which different cells and tissues acquire different programs of gene expression. From this point of view, genomic imprinting is a particular type of epigenetic regulation in which the activity of a gene is modified depending on the sex of the transmitting parent. Imprinting requires three main steps: gamete DNA marking, maintenance of the marking in embryo and adult somatic tissues, and resetting of marking at the beginning of gametogenesis [7]. In mammalian embryos there are two major cycles of epigenetic reprogramming of the genome: during preimplantation and during germ cell differentiation. These events have been widely studied in mouse embryos. After fertilization, the paternal and maternal genomes undergo a genomewide demethylation, followed by de novo methylation around the time of implantation. Imprinted alleles are protected from this wave of demethylation and

Prader-Willi and Angelman Syndromes

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remethylation to maintain their proper dosage effects. The second step in epigenetic reprogramming is necessary to reset imprinting in germ cells. This process takes place in primordial germ cells and consists of DNA demethylation, involving also imprinted genes, and subsequent remethylation according to the sex, so that in ovocytes all imprinted genes will have a maternal mark and in sperm cells a paternal one [8]. Occasionally, epigenetic information appears to be inherited through the germ line, and this is likely to be a result of incomplete erasure in the germ line (or after fertilization) [9].

Paradigm of Prader-Willi and Angelman Syndromes: Imprinting Regulation and Evolutionary Aspects

Deletions, duplication, mutations or alterations of imprinting of the only active allele, as well as uniparental disomy or loss of imprinting of the inactive allele lead to an unbalance (loss of function or gain of function) in the dosage of the gene product and may have phenotypic consequences. The first clinical syndromes recognized in humans as a result of anomalies at imprinted loci were Prader-Willi (PWS) and Angelman syndromes (AS) in 1989 [10]. PWS and AS result from loss of paternal or maternal contribution of the same genetic region at 15q11.q13 (PWS-AS imprinting domain). The PWS-AS imprinting domain has a bipartite structure, revealed by the smallest region of overlap in AS and PWS patients due to an imprinting center deletion. The PWS imprinting center (PWS-IC) is located in exon 1 of SNRPN and is defined by a cluster of CpG sites that are not methylated on the paternal allele and methylated on the maternal allele. The AS imprinting center (AS-IC) resides 35–40 kb upstream of SNRPN exon 1 and its proposed function is to promote the methylation of the adjacent PWS-IC in the maternal germline. This IC regulates the expression of several paternally expressed genes (among those SNRPN, MAGEL2, MKRN3, NDN and more than seventy snoRNAs) and two maternally expressed genes (UBE3A and ATP10C) (fig. 1) [11]. Proximal to this region the non-imprinted P gene is also mapped, encoding a tyrosine transporter whose deficiency contributes to the skin and ocular hypopigmentation that occurs in PWS and AS patients with deletion [12].The AS phenotype is caused by several genetic mechanisms leading to loss of function of the UBE3A gene, which encodes for an ubiquitin protein ligase, E6-AP, and is expressed only by the maternal allele in certain brain regions [13]. For PWS, instead, the phenotype seems to result from loss of function of a number of paternally expressed genes on chromosome 15q11-q13 [14]. In an evolutionary perspective, the PWS-AS domain was constructed relatively recently, between 180 and 105 million years ago. In marsupials, in which the first evidence of imprinting was found, the PWS-AS domain is absent, SNRPN and UBE3A are not on the same chromosome and a completely different gene, CNGA3 (now located on human chromosome 2) is situated in place of SNRPN, ~60 kb distant from UBE3A. The construction of the

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P1 0 AT

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26.1 26.2 26.3

25

24

15 21.1 21.2 21.3 22.1 22.2 22.3 23

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13 12

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M

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SN RP N

ND N

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Fig. 1. The PWS-AS imprinting domain. The imprinting center (IC) has bipartite structure: PWS-IC and AS-IC. On the maternal allele AS-IC confers methylation (M) of PWS-IC. As a result, all paternally expressed genes are silenced (empty boxes) and UBE3A and ATP10 are active (full boxes, arrows indicate transcription sense). On the paternal allele, PWS-IC remains unmethylated and activates all paternally expressed genes (e.g. MAGEL2, NDN, SNRPN).

PWS-AS domain was reached in eutherian through translocation of SNRPN beside UBE3A and insertion of miRNAs, snoRNAs and retroposed genes from all over the genome. Imprinting arose during or after this complex rearrangement (fig. 2) [15].

Prader-Willi Syndrome

This syndrome was clinically described by Prader and colleagues in 1956, but only in 1989 were the molecular bases of PWS and AS unraveled and related to an imprinting disorder [10]. A recent epidemiological study in the United Kingdom estimates an incidence of ~1 in 25,000 births and a prevalence of ~1 in 50,000 [16]. PWS is characterized by pre- and postnatal hypotonia, with a history of decreased fetal movements, poor suck and early failure to thrive. The fetus is usually podalic and delivery is commonly after term and requires cesarean section. Polyhydramnios is highly frequent. The hypotonia is most severe in early infancy, but improves somewhat over time. In the neonatal period feeble reflexes, lethargy and weak cry are often present. Feeding is usually an issue in the first few months of life and gavage is frequently required. Minor anomalies are often described and the typical facial appearance shows almond-shaped palpebral fissures, narrow bifrontal diameter, narrow nasal

Prader-Willi and Angelman Syndromes

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Eutheria 180 MYA

Mouse

210 MYA

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105 MYA Lactation

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Fig. 2. Evolution of imprinting in mammals. Imprinting is not present in non-mammals nor in monotremes, the most primitive mammals, which are capable of lactation but are still oviparous. The first evidence of imprinting has been found in marsupials. Modified from Hore et al. [15].

bridge and downturned mouth with thin upper lip, but not everyone has these features. Most patients have strabismus early in life. Frequently noted is also acromicria of the hands and feet with tapering fingers. Obesity is a hallmark of this syndrome, it occurs between the ages of 1 and 6 years and is due to hyperphagia. It is the major cause of morbidity and mortality in PWS and medical problems related to obesity are cardiopulmonary issues, increased risk for type II diabetes, obstructive sleep apnea and gastrointestinal complications [17, 18]. Hypoplastic genitalia, with cryptorchidism, scrotal hypoplasia and small penis in males and hypoplastic labia minora and clitoris in females are related to hypothalamic hypogonadism. An incomplete pubertal development is usually present, menarche may occur late and some female may have oligoamenorrhea or amenorrhea. Short stature is common and growth hormone (GH) deficiency has been demonstrated in most patients. Therapy with GH results in significant improvement of height and body composition (decreased fat mass and increased lean body mass) [17]. However, a significant number of deaths in children treated with GH has been reported, raising some concern about the safety of GH treatment in this population. A recent study shows that the major cause of death in children with PWS, who received GH treatment or did not, is a respiratory disease and no difference is found between the two groups. Nevertheless, since most deaths

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Clinical features and genetic mechanisms AS Loss of maternal contribution

PWS Loss of paternal contribution

• Mild- to moderate mental retardation • Hypotonia • Obesity • Hypogonadism • Short stature

• Severe mental retardation • Seizures • Ataxic movements • Absent speech • Inappropriate laughter

70%

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70%

25% maternal

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?

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10–15% UBE3A

⬍1%

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⬍1%

Unknown (all diagnostic test negative)

10%

Fig. 3. Clinical features and genetic mechanisms of PWS and AS. Modified from Cassidy et al. [14]. Pictures of PWS and AS patients: courtesy of Prof. Giovanni Neri.

occurred in the first 9 months of therapy, a possible aggravating role of GH at the start of treatment cannot be excluded [18]. The incidence of spinal deformity in PWS is high: scoliosis can occur at any age and kyphosis usually develops in early adulthood. Psychomotor development is mildly affected: walking is achieved at around 24 months and language development at 3–4 years. Mental retardation is in the mildto-moderate range and most patients have an IQ ranging from 60 to 70. Many show an unusual skill in doing jigsaw puzzles, suggesting a particular attention to visual details. Behavioral problems are frequent and include not only food-related problems (excessive appetite, absent sense of satisfaction, obsession with eating), but also temper tantrums, impulsivity, sleep disturbance, skin picking, obsessive-compulsive symptoms and anxiety [14, 17]. The genetic abnormality of PWS is the lack of expression of paternally inherited genes in chromosome 15q11-q13. Approximately 70% of affected individuals have a cytogenetically small deletion of chromosome segment 15q11-q13. Maternal uniparental disomy (UPD) accounts for 25%, imprinting defect (small deletion or mutation) for 2–5% and the remaining <1% is due to chromosomal rearrangement involving proximal 15q (fig. 3). Patients with UPD or imprinting defect generally have a milder phenotype than those with deletions [14]. The differential diagnosis for PWS in infancy includes many causes of neonatal hypotonia,

Prader-Willi and Angelman Syndromes

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particularly neuromuscular disorders. In childhood, syndromes presenting with mental retardation and obesity should be taken into account (Bardet-Biedl syndrome, Cohen syndrome and Albright hereditary osteodystrophy) [19].

Angelman Syndrome

The disorder, first described in 1965 by Angelman, has an estimated incidence between 1 in 10,000 and 1 in 40,000. The main clinical features are learning disability, ataxic and jerky movements, seizures, absent speech and happy, sociable disposition. Severe developmental delay and absent speech (fewer than 6 words) are seen in almost all patients. Most individuals are able to walk independently at between 2.5 and 5 years, with a characteristic jerky, stiff gait with upraised arms resembling a puppet-like gait; however, few patients remain nonambulatory. In spite of the severe speech impairment, many can communicate in other ways, such as sign language [20]. Birth growth parameters are normal, but frequently there is a slow down in growth of head circumference, usually resulting in microcephaly by age of 2 years. Facial dysmorphisms include deep-set eyes, large mouth with protruding tongue, widely spaced teeth and prominent chin. Skin hypopigmentation may be present. Seizures, varying from major motor to akinetic, usually begin before 3 years of age and can be severe, but many patients show improvement with age. Typical electroencephalographic patterns are described in AS and may suggest this diagnosis. Behavioral features of patients are sociable attitude, frequent laughter, hyperactivity, stereotypies such as hand flapping or twirling, attraction to water and fascination for shiny objects (mirror or plastic). In infancy, feeding problems are reported. Patients may have sleep disturbance with decreased need for sleep. In adult life, obesity and scoliosis develop frequently [20, 21]. AS is caused by a variety of genetic mechanisms (fig. 3) affecting the chromosome region 15q11-q13 [14] and classification by molecular defect shows some correlation to the clinical phenotype [22]. Patients with maternal deletion (70%) generally have a more severe phenotype including microcephaly, seizures, motor difficulties and language impairment. Physical features are clearly recognized and hypopigmentation of skin and eyes is common. Patients with paternal UPD (3–5%) show a milder phenotype: they have better physical growth, ataxia is less frequent and seizures have a lower prevalence. Facial features are more subtle and communication skills are better than in the deletion group. Patients with imprinting defect (2–5%) rarely have microcephaly and seizures are less frequent. They have a relatively better developmental and language ability, especially those who are mosaic for the imprinting defect. Patients with UBE3A mutation show generally some typical facial features and a smaller head size. Seizures are present in 50% of the patients. Compared to the deletion group, ataxia is less frequent, and development and language ability can be better. The differential diagnoses for AS include other genetic syndromes with mental retardation, absence of speech and seizures: Rett syndrome, 22q13.3 terminal deletion,

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Mowat-Wilson syndrome, alpha-thalassemia X-linked mental retardation (ATR-X) syndrome, Pitt-Hopkins syndrome and X-linked AS-like [23–25].

Genetic Counseling

It is currently crucial to identify the specific genetic mechanism that leads to PWS or AS in a given patient. This is not in the interest of phenotype-genotype correlation, but rather for the sake of counseling the parents about risk of recurrence. In fact, whereas deletions and uniparental disomies are sporadic events and therefore imply a low recurrence risk, imprinting defects and maternally inherited UBE3A mutation on the other hand have a 50% recurrence risk [14].

References 1 Linder D, McCaw BK, Hecht F: Parthenogenic origin of benign ovarian teratomas. N Engl J Med 1975; 292:63–66. 2 Kajii T, Ohama K: Androgenetic origin of hydatidiform mole. Nature 1977;268:633–634. 3 McGrath J, Solter D: Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 1984;37:179–183. 4 Cattanach BM, Kirk M: Differential activity of maternally and paternally derived chromosome regions in mice. Nature 1985;315:496–498. 5 Slack JM: Conrad Hal Waddington: the last Renaissance biologist? Nat Rev Genet 2002;3:889– 895. 6 Holliday R: Epigenetics: a historical overview. Epigenetics 2006;1:76–80. 7 Hall JG: Genomic imprinting: review and relevance to human diseases. Am J Hum Genet 1990;46:857– 873. 8 Reik W, Dean W, Walter J : Epigenetic reprogramming in mammalian development. Science 2001; 293:1089–1093. 9 Morgan HD, Sutherland HG, Martin DI, Whitelaw E: Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 1999;23:314–318. 10 Nicholls RD, Knoll JH, Butler MG, Karam S, Lalande M : Genetic imprinting suggested by maternal heterodisomy in nondeletion Prader-Willi syndrome. Nature 1989;342:281–285. 11 Shemer R, Hershko AY, Perk J, Mostoslavsky R, Tsuberi B, Cedar H, Buiting K, Razin A: The imprinting box of the Prader-Willi/Angelman syndrome domain. Nat Genet 2000;26:440–443.

Prader-Willi and Angelman Syndromes

12 King RA, Wiesner GL, Townsend D, White JG: Hypopigmentation in Angelman syndrome. Am J Med Genet 1993;46:40–44. 13 Kishino T, Lalande M, Wagstaff J: UBE3A/E6-AP mutations cause Angelman syndrome. Nat Genet 1997;15:70–73. Erratum in: Nat Genet 1997;15:411. 14 Cassidy SB, Dykens E, Williams CA: Prader-Willi and Angelman syndromes: sister imprinted disorders. Am J Med Genet 2000;97:136–146. 15 Hore TA, Rapkins RW, Graves JA: Construction and evolution of imprinted loci in mammals. Trends Genet 2007;23:440–448. 16 Whittington JE, Holland AJ, Webb T, Butler J, Clarke D, Boer H: Population prevalence and estimated birth incidence and mortality rate for people with Prader-Willi syndrome in one UK Health Region. J Med Genet 2001;38:792–798. 17 Cassidy SB: Prader-Willi syndrome. J Med Genet 1997;34:917–923. 18 Tauber M, Diene G, Molinas C, Hébert M: Review of 64 cases of death in children with Prader-Willi syndrome (PWS). Am J Med Genet 2008;146A:881– 887. 19 Gunay-Aygun M, Cassidy SB, Nicholls RD: PraderWilli and other syndromes associated with obesity and mental retardation. Behav Genet 1997;27:307– 324. 20 Clayton-Smith J, Laan L: Angelman syndrome: a review of the clinical and genetic aspects. J Med Genet 2003;40:87–95. 21 Williams CA, Beaudet AL, Clayton-Smith J, Knoll JH, Kyllerman M, Laan LA, Magenis RE, Moncla A, Schinzel AA, Summers JA, Wagstaff J: Angelman syndrome 2005: updated consensus for diagnostic criteria. Am J Med Genet 2006;140A:413–418.

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22 Lossie AC, Whitney MM, Amidon D, Dong HJ, Chen P, Theriaque D, Hutson A, Nicholls RD, Zori RT, Williams CA, Driscoll DJ: Distinct phenotypes distinguish the molecular classes of Angelman syndrome. J Med Genet 2001;38:834–845. 23 Williams CA, Lossie A, Driscoll D : Angelman syndrome: mimicking conditions and phenotypes. Am J Med Genet 2001;101:59–64. 24 Zweier C, Peippo MM, Hoyer J, et al: Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (PittHopkins syndrome). Am J Hum Genet 2007;80: 994–1001.

25 Gilfillan GD, Selmer KK, Roxrud I, et al: SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome. Am J Hum Genet 2008;82:1003–1010.

Fiorella Gurrieri, MD Istituto di Genetica Medica Università Cattolica del Sacro Cuore Largo F. Vito, 1, IT–00168 Roma (Italy) Tel. +39 0630 154 927; Fax +39 0635 0031, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 29–37

Muscle Involvement and IGF-1 Signaling in Genetic Disorders: New Therapeutic Approaches Laura Barberi ⭈ Gabriella Dobrowolny ⭈ Laura Pelosi ⭈ Cristina Giacinti ⭈ Antonio Musarò Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Histology and Medical Embryology, CE-BEMM, and Interuniversity Institute of Myology, Sapienza University of Rome, Rome, Italy

Abstract In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of muscle diseases. With the characterization of mutations responsible for muscle dysfunction in several inherited pathologies, and the identification of novel signaling pathways, subtle alterations in which can lead to significant defects in muscle metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress has been slow in therapeutic applications of our newly gained knowledge. The complexity of muscle types, the intimate relationship between structural integrity and mechanical function, and the sensitivity of skeletal muscle to metabolic perturbations have impeded rapid progress in successful clinical intervention. The relatively poor regenerative properties of striated muscle compound the devastating effects of muscle degeneration. Perhaps the most difficult hurdle is the sheer volume of tissue that must be treated to effect a significant improvement in quality of life. Recent studies on the role of insulin-like growth factor–1 in skeletal muscle growth and homeostasis have excited new interest in this important mediator of anabolic pathways and suggest promising new avenues for intervention in catabolic disease. In this review, we will discuss the potential therapeutic role of local insulin-like growth factor 1 in the treatment of muscle wasting associated with muscle Copyright © 2009 S. Karger AG, Basel diseases.

Although the genetic defects responsible for muscle dysfunction in several inherited pathologies have been well characterized, the molecular basis of muscle wasting remains elusive. It is generally accepted that the primary cause of functional impairment in muscle is a cumulative failure to repair damage related to an overall decrease in anabolic processes. In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of muscle diseases. However, the complexity of muscle

types, the intimate relationship between structural integrity and mechanical function, and the sensitivity of skeletal muscle to metabolic perturbations have impeded rapid progress in successful clinical intervention. In addition, the relatively poor regenerative properties of striated muscle compound the devastating effects of muscle degeneration. In this context, where direct therapeutic approaches to redress the primary disease are still suboptimal, it may be more effective to focus on strategies for improving skeletal muscle function. In this review we will discuss the potential therapeutic role of insulin-like growth factor 1 (IGF-1) in the treatment of muscle wasting associated with several muscle diseases.

Molecular Complexities of IGF-1 Transcription

IGF-1 controls growth and metabolism in several organs and tissues during both embryonic and postnatal development [1, 2]. At the structural level, IGF-1 is related to insulin with which it shares a 50% amino acid identity. Unlike the insulin gene, the IGF-1 gene locus encodes multiple proteins with variable N-terminal and C-terminal amino acid sequences. The amino acid sequence of the mature peptide differs from that of insulin by retention of the C peptide, by a short extension of the A chain to include a novel domain D, and by the presence of variable C-terminal E peptides (fig. 1). Although the IGF-1 gene is highly conserved in numerous species, its relatively large size (over 70 kb), combined with complex transcriptional and splicing patterns, has complicated its analysis. The primary structure of IGF-1 is highly conserved in placental mammalian species: canine [3], bovine [4], ovine [5], porcine [6] and human [7] IGF-Is are identical, whereas rat [8] and mouse [9] IGF-1s differ from human by 3 and 4 amino acids, respectively, and there is only one amino acid difference between these rodent IGF-1s. The rodent IGF-1 gene contains six exons, separated by five introns (fig. 1). Exons 1 and 2 encode distinct 5´UTRs, as well as different parts of the signal peptide, and are therefore termed leader exons. Exon 3 encodes 27 amino acids that are part of the signal peptide and common to all isoforms, as well as part of the mature IGF-1 peptide. Exon 4 encodes the rest of the mature peptide and 16 amino acids of the aminoterminal region of the E-peptide, which is also common to all IGF-1 mRNAs. Exons 5 and 6 encode two distinct carboxy-terminal E-peptides and the 3´UTR. Two promoters initiating at alternate 5⬘exons add further complexity. Although IGF-1 transcripts are not exclusively tissue-restricted, those that initiate at exon 2 predominate in the liver, are highly growth hormone responsive and as such are major endocrine effectors of GH [10]. By contrast, transcripts initiating at exon 1 are widely

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Exon 1

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Exon 6

D Eb/MGF Ea

Fig. 1. Schematic representation of a rodent IGF-1 gene. The rodent IGF-1 gene contains six exons (filled boxes), separated by five introns (dashes). Both exons 1 and 2 contain multiple transcription start sites (horizontal arrows). Translation initiation codons (AUG) are located at exons 1, 2 and 3 (vertical arrows). These exons code for the signal peptide of precursor IGF-1. Exons 5 and 6 each encode distinct portions of the E peptides.

expressed in all tissues, and are less affected by circulating growth hormone levels, presumably performing autocrine or paracrine functions. The alternate splicing at the 5⬘ ends of these two IGF-1 transcripts generates different signal peptides, which purportedly affects the precise N-terminal pro-peptide cleavage site [10, 11]. The functions of the proteins encoded by these different transcripts are widely debated but a cohesive picture has yet to emerge. Elucidation of isoform function is also complicated by alternate splicing at the 3⬘ end of IGF-1 transcripts. This produces variability in the length and amino acid sequence of the E peptide, and in the length and base sequence of the 3⬘UTR. To date, two different splice patterns have been documented in rodents (fig. 1). Each generates E peptides with a common N-terminal 16 aa sequence, and alternate C-terminal sequences [10–13]. If exon 4 splices to exon 6 (the predominant pattern), the length of the 3⬘UTR is highly variable, but in all cases the Ea peptide is generated with 19 additional amino acids. If exon 4 splices to exons 5 and 6, a variant known as Eb is encoded, which is frameshifted relative to exon 6 and therefore a different 25 aa sequence is added to the common 16 aa encoded by exon 4. Although E peptide choice appears to be independent of promoter use, Eb-containing transcripts are more abundant in liver, whereas Ea-containing transcripts are widespread in extrahepatic tissues. In addition, the analysis of the amino acid structure of both E peptides has revealed the presence of two N-linked glycosylation sites only in the Ea peptide, but not in the Eb peptide, suggesting that this post-translational modification is involved in a biological action of the IGF-1 isoform [14]. The IGF-1Eb isoform is also upregulated in muscles subjected to stretch and has been named mechano growth factor (MGF) [13]. It has been also reported that, unlike mature IGF-1, the distinct E domain of MGF inhibits terminal differentiation whilst increasing myoblast proliferation [15]. The determination of E peptide function and fate awaits the availability of epitope-specific antibodies, since it is unclear when or whether E peptides are cleaved from the mature IGF-1 protein. Notably, E peptide

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splicing patterns are different in the human gene [16], an anomaly that will need to be considered in the future when translating the results of animal research into clinical applications. In humans, three mRNA variants with alternatively spliced 3’-end have been identified [17]. Similarly to rat, splicing of human exon 4 to exon 6 yields an mRNA sequence which encodes the 35 amino acid Ea peptide [18], which shares 91% homology with the mouse Ea peptide [9]. The second mRNA splice variant identified in human liver is an exon 4–5 splice variant, which encodes an extension peptide termed Eb peptide [19, 20]. Human Eb peptide contains additional 61 amino acids encoded by exon 5 [20], giving rise to an E peptide with the total length of 77 amino acids. The third mRNA splice variant identified in human liver contains exon 4, 49 bp of exon 5 and then exon 6 (exons 4–5–6) and yields an extension peptide with a total predicted length of 41 amino acids, termed Ec peptide [21]. This human Ec peptide shares 73% homology with rat Eb peptide (MGF) and is considered to be its counterpart [21]. Possible existence of the exon 4–5 splice variant, which codes for Eb peptide in humans, was also predicted in rats [8].

Importance of IGF-1 Isoforms

Analyses of transgenic mice expressing different IGF-1 isoforms have provided insight into the role of local IGF-1 signaling in the physiology of striated muscle [22]. By controlling the transcription of IGF-1 transgenes with different promoters, it has been possible to characterize the role of the local and/or circulating form of IGF-1 on muscle cell and tissue function [22]. The fact that IGF-1 can act either as a circulating hormone or as a local growth factor has confounded previous analyses of animal models in which transgenic IGF-1 synthesized in extrahepatic tissues was released into the circulation [23, 24]. Overexpression of one IGF-1 isoform in the heart prevented activation of cell death in the viable myocardium after infarction, limiting ventricular dilation, myocardial loading, cardiac hypertrophy, and diabetic cardiomyopathy, supporting the notion that constitutive over-expression of IGF-1 in cardiomyocytes protects them from apoptosis and hypertrophy in the normal and pathological heart [23]. In another study, overexpression of a different IGF-1transgene in the heart induced physiological cardiac hypertrophy that progressed to maladaptive hypertrophy [25]. The transgenic IGF-1 model generated in this study demonstrated that short-term systolic performance benefit of increased IGF-1, but ultimately it diminishes systolic performance raising doubt about the therapeutic value of chronic IGF-1 administration. The discrepancies in these phenotypes underscore the normal physiological difference between IGF-1 isoform function. Promising a survival factor as IGF-1 might be, substantial evidence supports its involvement in mitogenesis and neoplastic transformation [26], suggesting that this signaling pathway plays an important role in the

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process of tumor promotion. The neoplastic potential of at least certain IGF-1 isoforms is an obvious concern to be taken into account when designing IGF-therapeutic strategies for human pathologies, where the specific role of each IGF-1 isoform must be viewed in the appropriate tissue context.

Effects of the Local Isoform of IGF-1 on Muscle Homeostasis and Regeneration

The prolongation of skeletal muscle strength in aging and neuromuscular disease has been the objective of numerous studies employing a variety of approaches. IGF-1, involved in muscle growth and hypertrophy, decline during postnatal life, raising the prospect that this decline contributes to the progress of muscle atrophy in senescence, and limits the ability of skeletal muscle tissue to effect repair or to regenerate. To test this possibility we generated a transgenic mouse in which the local isoform of IGF-1 (mIGF-1) is driven by MLC promoter (MLC/mIGF-1) [27]. The MLC regulatory elements included in this construct activate linked gene expression as early as E9.5 days in embryonic mouse development, and expression continues to be high in the fastest type IIb fibers. Transgenic animals exhibits marked skeletal muscle hypertrophy and no undesirable side effects such as tumor formation, as revealed in transgenic mice over-expressing the circulating IGF-1 isoform. The importance of appropriate IGF-1 isoform selection is further underscored by preliminary analysis of mouse lines generated with a second IGF-1 transgene. The IGF-1 isoform used (cIGF-1) differed from the mIGF-1 transgene only in a variant C-terminal peptide, which was presumably responsible for the dramatic phenotypic differences of cIGF-1 mice. These animals did not display pronounced muscle hypertrophy but had increased levels of circulating IGF-1, mild cardiac hypertrophy, an increased incidence of late onset neoplasia [unpubl. obs.]. Thus, the choice of isoform is critical to the design of gene therapeutic strategies employing IGF-1. The increased muscle mass in mIGF-1 transgenic mice was associated with augmented force generation compared to age-matched wild-type littermates. Overexpression of the mIGF-1 transgene also promoted and preserved the regenerative capacity of muscle tissues and the ability to repair of damaged muscle during aging [27]. The anabolic effects of IGF-1 may be due in part to stimulation of activation of satellite cells [22]. It is not known whether in MLC/mIGF-1 transgenic animals, the satellite cells have an increased ability for self-renewal or whether there is an increased recruitment of nonsatellite cells. Our recent experimental evidence indicates that mIGF-1 promotes the two suggested pathways, which can be considered two temporally separated events of the same biological process.

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We demonstrated that upon muscle injury, stem cells expressing c-Kit, Sca-1, and CD45 antigens increased locally and the percentage of the recruited cells were conspicuously enhanced by mIGF-1 expression [28]. FACS profiles of tissues from wild-type and MLC/mIGF-1 transgenic mice, whose muscles were injured with cardiotoxin, revealed a consistent increase of side population (SP) cells in the bone marrow, compared to noninjured controls whose percentages increase in MLC/mIGF-1 transgenic mice [28]. Thus, humoral signals emanating from the injured muscles were sufficient to induce stem cell proliferation in the bone marrow, but not in other tissues. One of the crucial parameters of tissue regeneration is the microenvironment in which the stem cell population should operate. Stem cell microenvironment, or niche, provides essential cues that regulate stem cell proliferation and that direct cell fate decisions and survival. Moreover, loss of control over these cell fate decisions might lead to cellular transformation and cancer. More recently, we investigated whether accelerating the course of muscle repair through mIGF-1 expression involves modulation of the inflammatory response, which represents one of the critical stages of tissue regeneration [29]. We show that local expression of mIGF-1 transgene accelerates the regenerative process of injured skeletal muscle, modulating the inflammatory response, and limiting fibrosis [29]. At the molecular level, mIGF-1 expression significantly downregulated proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and modulated the expression of CC chemokines involved in the recruitment of monocytes/macrophages [29]. Analysis of the underlying molecular mechanisms revealed that mIGF-1 expression modulated key players of inflammatory response, such as macrophage migration inhibitory factor (MIF), high mobility group protein-1 (HMGB1), and transcription NF-κB [29]. The rapid restoration of injured mIGF-1 transgenic muscle was also associated with connective tissue remodeling and a rapid recovery of functional properties [29]. By modulating the inflammatory response and reducing fibrosis, supplemental mIGF-1 creates a qualitatively different environment for sustaining more efficient muscle regeneration and repair. Thus, mIGF-1 can overcome the normal inability of skeletal muscle to sustain regeneration and repair and as such represents a potentially effective gene therapeutic strategy to combat muscle wasting.

Effects of mIGF-1 on Muscular Dystrophy and Muscle Wasting

Because it is clear that mIGF-1 can prevent aging- related loss of muscle function [28, 22], it is possible that mIGF-1 can prevent or diminish muscle loss associated with disease.

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To prove this hypothesis, we introduced mIGF-1 into the mdx dystrophic animals (mdx/mIGF-1) [30]. This approach allowed for the assessment of the maximum potential benefit that could be derived from IGF-I expression for dystrophic muscle, as well as examination of both the diaphragm and the extensor digitorum longus (EDL), which display a spectrum of dystrophic pathologies. By analyzing both muscle morphology and function in transgenic mdx/mIGF-1, we observed a significant improvement in muscle mass and strength, a decrease in myonecrosis, and a reduction in fibrosis in aged diaphragms [30]. In particular, even though IGF-1 has been shown to stimulate fibroblasts, there was a net decrease in fibrosis in diaphragms of the mdx/mIGF-1 mice [30]. In fact, age-related fibrosis in the mdx diaphragm was effectively eliminated by mIGF-1 expression. It may be that the efficient and rapid repair of the mdx/mIGF-1 muscles prevents the establishment of an environment into which the fibroblasts migrate. This is of particular relevance to the human dystrophic condition where virtually all skeletal muscles succumb to fibrosis. Thus, the results found in the mouse diaphragm suggest that mIGF-1 might be effective not only in increasing muscle mass and strength, but also in reducing fibrosis associated with the disease. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin. More recently, Abmayr et al. [31] demonstrated that co-injection of the rAAVmicrodystrophin and rAAV-mIGF-1 vectors resulted in increased muscle mass and strength, reduced myofiber degeneration, and increased protection against contraction-induced injury. These results suggest that a dual-gene, combinatorial strategy could enhance the efficacy of gene therapy of DMD and underscored the importance of rAAV vectors due to their relative lack of immunologic and toxic side effect combined with their potential for body-wide systemic gene delivery to muscle [31]. Recent studies on the role of mIGF-1 in skeletal muscle growth and homeostasis have excited new interest in this important mediator of anabolic pathways and suggest promising new avenues for intervention in different catabolic diseases. Indeed, it has been recently reported that transgenic overexpression of mIGF-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction (CLVD) [32] and counteracts the symptoms of amyotrophic lateral sclerosis (ALS) [33, 34], a progressive, lethal neuromuscular disease associated with the degeneration of motor neurons, leading to muscle atrophy and paralysis [34]. In particular, we demonstrated that mIGF-1 induces satellite cell activity, stabilizes neuromuscular junctions and leads to a reduction in astrocytosis in the spinal cord of ALS mouse model [33, 34].

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Conclusions

Although the evidence to date for growth factor supplementation is encouraging, it remains to be determined which IGF-1 isoforms or combination of growth factors elicit the most effective response. These preliminary studies provide exciting avenues for future discovery, however true innovation in this field will undoubtedly derive from the integration of our insights with other key advances in regenerative research, to achieve precise modulation of the regenerative response, and to form a cohesive and coherent strategy that addresses the short-term, medium and long-term aspects of the therapeutic process.

Acknowledgments Work in the authors’ laboratories has been supported by Italian Telethon, MDA, AIRC, ASI, AFM and Progetti Ricerca Università.

References 1

2

3

4

5

6

7

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Stewart CE, Rotwein P: Growth, differentiation, and survival: multiple physiological functions for insulinlike growth factors. Physiol Rev 1996;76:1005–1026. Butler AA, LeRoith D: tissue-specific versus generalized gene targeting of the igf1 and igf1r genes and their roles in insulin-like growth factor physiology. Endocrinology 2001;142:1685–1688. Delafontaine P, Lou H, Harrison DG, Bernstein KE: Sequence of a cDNA encoding dog insulin-like growth factor I. Gene 1993;130:305–306. Francis GL, Upton FM, Ballard FJ, McNeil KA, Wallace JC: Insulin-like growth factors I and II in bovine colostrums: sequences and biological activities compared with those of a potent truncated form. Biochem J 1988;251:95–103. Francis GL, McNeil KA, Wallace JC, Ballard FJ, Owens PC: Sheep insulin-like growth factors I and II: sequences, activities and assays. Endocrinology 1989;124:1173–1183. Francis GL, Owens PC, McNeil KA, Wallace JC, Ballard FJ: Purification, amino acid sequences and assay crossreactivities of porcine insulin-like growth factor-I and –II. J Endocrinol 1989;122:681–687. Rinderknecht E, Humbel RE: The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 1978;253:2769–2776. Shimatsu A, Rotwein P: Mosaic evolution of the insulin-like growth factors. Organization, sequence, and expression of the rat insulin-like growth factor I gene. J Biol Chem 1987;262:7894–7900.

9 Bell GI, Stempien MM, Fong NM, Rall LB: Sequences of liver cDNAs encoding two different mouse insulin-like growth factor I precursors. Nucleic Acid Res 1986;14:7873–7882. 10 LeRoith D, Roberts CT Jr: Insulin-like growth factor I (IGF-I): a molecular basis for endocrine versus local action? Mol Cell Endocrinol 1991;77:C57–61. 11 Adamo ML, Ben-Hur H, Roberts CT Jr, LeRoith D: Regulation of start site usage in the leader exons of the rat insulin-like growth factor-I gene by development, fasting, and diabetes. Mol Endocrinol 1991;5: 1677–1686. 12 LeRoith D, Roberts, CT Jr: Insulin-like growth factors. Ann NY Acad Sci 1993;692:1–9. 13 McKoy G, Ashley W, Mander J, Yang SY, Williams N, Russell B, Goldspink G: Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation. J Physiol 1999;516:583–592. 14 Bach MA, Roberts CT Jr, Smith EP, LeRoith D: Alternative splicing produces messenger RNAs encoding insulin-like growth factor-I prohormones that are differentially glycosylated in vitro. Mol Endocrinol 1990;4:899–904. 15 Yang SY, Goldspink G: Different roles of the IGF-I Ec peptide (MGF) and mature IGF-I in myoblast proliferation and differentiation. FEBS Lett 2002; 522:156–160. 16 Adamo ML, Neuenschwander S, LeRoith D, Roberts CT Jr: Structure, expression, and regulation of the IGF-I gene. Adv Exp Med Biol 1993;343:1–11.

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17 Shavlakadze T, Winn N, Rosenthal N, Grounds MD: Reconciling data from transgenic mice that overexpress IGF-I specifically in skeletal muscle. Growth Horm IGF Res 2005;15:4–18. 18 Jansen M, van Schaik FM, Ricker AT, Bullock B, Woods DE, Gabbay KH, Nussbaum AL, Sussenbach JS, Van den Brande JL: Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature 1983;306:609–611. 19 Rotwein P, Pollock KM, Didier DK, Krivi GG: Organization and sequence of the human insulinlike growth factor I gene. J Biol Chem 1986;261: 4828–4832. 20 Rotwein P: Two insulin-like growth factor I messenger RNAs are expressed in human liver, Proc Natl Acad Sci USA 1986;83:77–81. 21 Chew SL, Lavender P, Clark AJ, Ross RJ: An alternatively spliced human insulin-like growth factor-I transcript with hepatic tissue expression that diverts away from the mitogenic IBE1 peptide. Endocrinology 1995;136:1939–1944. 22 Musarò A, Rosenthal N: The critical role of insulinlike growth factor-1 isoforms in the physiopathology of skeletal muscle. Curr Genomics 2006;3: 19–32. 23 Delaughter MC, Taffet GE, Fiorotto ML, Entman ML, Schwartz RJ. Local insulin-like growth factor I expression induces physiologic, then pathologic, cardiac hypertrophy in transgenic mice. FASEB J 1999;13:1923–1929. 24 Li Q, Li B, Wang X, Leri A, Jana KP, Liu Y, Kajstura J, Baserga R, Anversa P: J Clin Invest 1997;100: 1991–1999. 25 Leri A, Liu Y, Wang X, Kajstura J, Malhotra A, Meggs LG, Anversa P: Overexpression of insulinlike growth factor-1 attenuates the myocyte reninangiotensin system in transgenic mice. Circ Res 1999;84:752–762. 26 Pollak MN, Schernhammer ES, Hankinson SE: Insulin-like growth factors and neoplasia. Nat Rev Cancer 2004;4:505–18.

27 Musarò A, McCullagh K, Paul A, Houghton L, Dobrowolny G, Molinaro M, Barton ER, Sweeney HL, Rosenthal N: Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle. Nat Genet 2001;27:195– 200. 28 Musarò A, Giacinti C, Borsellino G, Dobrowolny G, Pelosi L, Cairns L, Ottolenghi S, Bernardi G, Cossu G, Battistini L, Molinaro M, Rosenthal N: Muscle restricted expression of mIGF-1 enhances the recruitment of stem cells during muscle regeneration. Proc Natl Acad Sci USA 2004;101:1206–1210. 29 Pelosi L, Giacinti C, Nardis C, Borsellino G, Rizzuto E, Nicoletti C, Wannenes F, Battistini L, Rosenthal N, Molinaro M, Musarò A: Local expression of IGF-1 accelerates muscle regeneration by rapidly modulating inflammatory cytokines and chemokines. FASEB J 2007;21:1393–1402. 30 Barton ER, Morris L, Musaro A, Rosenthal N, and Sweeney HL: Muscle specific expression of Insulinlike Growth Factor I counters muscle decline in mdx mice. J Cell Biol 2002;157:137–147. 31 Abmayr S, Gregorevic P, Allen JM, Chamberlain JS: Phenotypic improvement of dystrophic muscles by rAAV/microdystrophin vectors is augmented by Igf1 codelivery. Mol Ther 2005;12:441–450. 32 Schulze PC, Fang J, Kassik KA, Gannon J, Cupesi M, MacGillivray C, Lee RT, Rosenthal N: Transgenic overexpression of locally acting insulin-like growth factor-1 inhibits ubiquitin-mediated muscle atrophy in chronic left-ventricular dysfunction. Circ Res 2005;97:418–426. 33 Dobrowolny G, Giacinti C, Pelosi L, Nicoletti C, Winn N, Barberi L, Molinaro M, Rosenthal N, Musarò A: Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model. J Cell Biol 2005;168:193–199. 34 Musarò A, Dobrowolny G, Rosenthal N: The neuroprotective effects of a locally acting IGF-1 isoform. Exp Gerontol 2007;42:76–80.

Antonio Musarò; MD Department of Histology and Medical Embryology Via A. Scarpa14 IT–00161 Rome (Italy) Tel. +39 0649 766 956, Fax +39 06 44 62 854, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 38–52

Mitochondrial Encephalomyopathies and Related Syndromes: Brief Review Enrico Bertini ⭈ Adele D’Amico Bambino Gesu’ Children’s Research Hospital, Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Disorders, Department of Laboratory Medicine, Rome, Italy

Abstract A brief and comprehensive review on mitochondrial cytopathies is reported showing the extreme clinical and genetic heterogeneity of these disorders. Syndromes of mitochondrial cytopathies encompass most of the medical specialties and diagnosis of mitochondrial cytopathies is complicated, needing the combination of multiple expertise: muscle morphology, neuroradiology, bioCopyright © 2009 S. Karger AG, Basel chemistry (enzymology, chemical analysis), and genetics.

Mitochondria have their own distinct DNA. Each mitochondrion contains several circular double-stranded DNA copies that are normally 16,569 bp in length. Each copy contains 37 genes that code for several respiratory chain structural proteins and for mitochondrial DNA transcription and translation (22 tRNAs) factors. Mitochondrial DNA abnormalities were first linked to human disease in 1988. In that year, Leber’s hereditary optic neuropathy and several progressive muscle disorders were found to be caused by mutations in mitochondrial DNA [1, 2]. Aerobic metabolism depends on the hundreds of mitochondria that every cell in the body contains. Cellular dysfunction results when the proportion of mutated mitochondrial DNA strands exceeds a threshold level. Because sperm do not contribute with mitochondria to the zygote, mutations in mitochondrial DNA are classically inherited only from the mother (maternal inheritance). Mitochondria have their own distinct DNA. The proportion of mutated mitochondria can differ widely from one oocyte to the other. Consequently, siblings can have widely varying symptoms and severity of disease. Similarly, the proportion of mutant mitochondrial DNA differs from cell to cell in an embryo, so different daughter cell lines can have widely varying levels of cell dysfunction. Slightly later it became evident that also defects in chromosomal DNA can also cause mitochondrial disease, because chromosomal DNA supplies most of the proteins necessary for mitochondrial DNA replication and expression. These conditions

were inherited by a Mendelian trait in an autosomal-dominant or autosomal-recessive fashion [3]. Describing an overview of mitochondrial encepahlomyopathies, we will keep with the concept that the term ‘mitochondrial encephalomyopathy’ or mitochondrial cytopathy (MC) refers to defects of oxidative phosphorylation of the mitochondrial respiratory chain, excluding other mitochondrial disorders related to other mitochondrial functions such as iron homeostasis, β-oxidation, etc. The clinical presentation in these disorders is extraordinarily heterogeneous, because any tissue can be affected, isolatedly or multisystemically. First of all, symptoms may begin at any age but the first symptoms are observed before 1 month of age in more than one third of the cases and before the age of 2 years in 80% of the cases [4]. Tissues that are highly energy dependent are more frequently affected, such as brain, skeletal muscle, heart and kidney. Skeletal muscle is the most often affected tissue, suggesting that these are somatic mutations, that is, spontaneous events that arose in myoblasts or in myoblast precursors after germ-layer differentiation. Pure myopathy, dominated by exercise intolerance with or without myoglobinuria, is generally highly evocative for a mitochondrial encephalomyopathy. Exercise intolerance is a common complaint, which, if the patient has no objective weakness, increased serum creatine kinase (CK) levels or abnormal electromyography (EMG), is often dismissed as ‘psychogenic’ or mislabeled as ‘chronic fatigue syndrome’ or ‘fibromyalgia rheumatica’. Many patients with mutations in mtDNA protein-coding genes fall into this group, and the lack of maternal inheritance further deflects the physician from thinking about a mitochondrial cytopathy. When faced with these puzzling patients, it is important to consider the possibility of a mitochondrial disease and to obtain, at least, a resting lactate value. Myoglobinuria, and especially recurrent myoglobinuria, is commonly associated with blocks in the utilization of the two major sources of energy for muscle contraction, glycogen or fatty acids [5]. Another special symptom that may occur in a mitochondrial encephalomyopathy is sideroblastic anemia or megaloblastic anemia generally associated with complex I deficiency. The fact that tissues other than muscle can be affected selectively suggests that we should keep an open mind about the possibility that somatic mutations of mtDNA protein-coding genes may be involved in other tissue-specific disorders such as cardiopathy or encephalopathy. A multisystemic presentation affecting multiple tissues is highly evocative of a mitochondrial encephalomyopathy; however, it is important that the clinician confirms the clinical suspect searching for persistent or fluctuating increased lactate in blood, urine and CSF or by MR spectroscopy. If lactate is not increased further work for molecular genetic confirmation is not recommended. The diagnostic approach of mitochondrial encephalomyopathies is complicated, and needs the combination of multiple expertise: muscle morphology, neuroradiology, biochemistry (enzymology, chemical analysis), and genetics.

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In muscle morphology, ragged-red fibers (RRF), the histochemical hallmark of massive mitochondrial proliferation in muscle, are typically seen in patients with mtDNA mutations that impair overall mitochondrial protein synthesis, such as mutations in tRNA or rRNA genes, single or multiple deletions, or mtDNA depletion (most frequent and known phenotypes are MELAS, MERFF, Pearson syndrome, KearnSayre syndrome (KSS), PEO). Conversely, RRF are absent in muscle biopsies from patients with mutations in mtDNA protein-coding genes, such as the NARP/MILS mutations in the ATPase 6 gene, or the various mutations in genes encoding complex 1 subunits (ND genes) associated with Leber’s hereditary optic neuropathy (LHON).

Genetics

Mitochondrial diseases can be due to mitochondrial DNA (mtDNA) mutations or mutations in nuclear genes. Mitochondrial DNA is more subjected to mutations because mtDNA is more susceptible to damage by ROS resulting from low efficiency of mtDNA repair systems, lack of protection by histones, and unique structural characteristics favoring mutations [6]. Point mutations in mitochondrial DNA (mtDNA) are generally transmitted by maternal inheritance; however, point mutations in protein-coding genes may arise de novo and cause sporadic disorders. Moreover, severe large-scale deletions of mtDNA rearrangements are rarely segregated through generations and frequently arise sporadically. With time, nuclear DNA mutations have been identified as being responsible for respiratory chain defects [6]. Two of the four most common causes of Leigh syndrome, a devastating neurodegenerative disease of infancy or childhood, are due to specific respiratory chain defects: complex I deficiency, and COX deficiency due to SURF-I deficiency [7, 8]. Both conditions are inherited as autosomal-recessive traits. The product of SURF-I is indeed a mítochondrial protein, which appears to act at the third stage in the four-step process of COX assembly. The other two common causes of Leigh syndrome are pyruvate dehydrogenase complex (PDHC) deficiency and the T8993G mutation in the mtDNA ATPase 6 gene. PDHC deficiency is usually inherited as an X-Iinked dominant trait while the T8993G mutation is the most common cause of maternally inherited Leigh syndrome (MILS). In addition to SURFI, other COX assembly genes were studied, and pathogenic mutations were found in the SC02 gene in 3 infants with fatal infantile cardiomyopathy and encephalopathy, but without the typical neuropathological features of Leigh syndrome [7]. The SC02 gene encodes a copper-binding protein that must play a crucial role in the assembly of COX, which contains two copper atoms. This essential function coupled with Northern blot provides evidence that the SC02 protein is expressed predominantly in heart and muscle and explains both the clinical phenotype and the extremely low levels of COX activity found in heart and muscle.

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A special group of Mendelian mitochondrial diseases reflects the gradual loss of autonomy of the mitochondrial genome, which now depends heavily on factors encoded by nuclear genes for some of its essential functions, including transcription, translation, and replication. Disorders of intergenomic signaling are due to mutations in nuclear genes that, directly or indirectly, control mtDNA number, function, or integrity. The first examples of such faulty ‘dialogue’ between the two genomes was offered by patients with autosomal-dominant progressive external ophthalmoplegia (PEO) and multiple mtDNA deletions in muscle (instead of the single type of mtDNA rearrangement that characterizes each patient with KSS or sporadic PEO) [7]. Another disorder of intergenomic communication, resulting in tissue-specific paucity of mtDNA copies (‘mtDNA depletion’), was described in infants with severe congenital myopathy or hepatopathy [3]. There are milder myopathic forms of mtDNA depletion and the clinical spectrum may involve both central and peripheral nervous systems. Several genetic defects have been characterized for conditions related to mtDNA depletion and others still remain elusive. A defect of polymerase-γ (POLG) has been reported in children with Alpers syndrome and mtDNA depletion, and in adults affected by neuropathy and ataxia inherited as an autosomal-recessive trait [9], and dominant polymerase-γ deficiency has also been found in adult patients with autosomal-dominant PEO. Another example of disorder of intergenomic signaling is an autosomal-recessive form of PEO known with the acronym of MNGIE, for mitochondrial neurogastrointestinal encephalomyopathy dominated by gastrointestinal problems (chronic diarrhea, intestinal pseudoobstruction) leading to cachexia and early death. The gene was characterized by Nishino et al. [10] in 1999 as thymidine phosphorylase (TP). TP is widely expressed in human tissues, including some that are selectively involved in MNGIE, such as the gastrointestinal system, brain, and peripheral nerves. Additional symptoms and signs include ptosis and ophthalmoplegia, peripheral neuropathy, and leukoencephalopathy. In contrast to the autosomal-dominant forms of PEO, which are largely confined to muscle, autosomal recessive PEO syndromes with multiple mtDNA deletions tend to be multisystemic [7]. Muscle biopsy shows COX-negative RRF, biochemical evidence of COX deficiency, and molecular evidence of mtDNA multiple deletions, sometimes associated with mtDNA depletion [7]. We will now summarize below the main clinical presentations of mitochondrial cytopathies (MC) dividing them systematically by the different tissues predominantly involved (table 1).

Syndromes in Mitochondrial Cytopathies

Myopathy Myopathy is a frequent feature in MC. It is responsible for muscle weakness with myalgia and exercise intolerance [7]. Sometimes rabdomyolysis can be the leading symptom.

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Table 1. Main symptoms of mitochondrial cytopathies Affected organ

Symptoms

Central nervous system

apnea, lethargy, hypotonia, coma in the neonatal period, hypotonia, psychomotor regression, cerebellar ataxia, stroke-like episodes, myoclonus, seizures, dementia, spasticity, headache, hemiparesis in infants and children, leukodystrophy, myoclonus

Muscle

myopathy, poor head control, limb weakness, myalgia, exercise intolerance, rhabdomyolysis

Liver

liver enlargement, hepatocellular dysfunction

Heart

cardiomyopathy, heart block

Kidney

proximal tubulopathy, nephrotic syndrome, renal failure, tubulointerstitial nephritis

Gut

vomiting, diarrhea, villous atrophy, colonic pseudo-obstruction, exocrine pancreas dysfunction

Endocrine

diabetes mellitus, growth hormone deficiency, hypoparathyroidism, hypothyroidism

Bone marrow

sideroblastic anemia, neutropenia, thrombocytopenia

Ear

hearing loss

Eye

progressive external ophthalmoplegia, pigment retinal degeneration, ptosis, diplopia, cataract

Skin

mottled pigmentation, discoloration, acrocyanosis, vitiligo, cutis marmorata, anhydrosis and jaundice; trichothiodystrophy, hirsutism alopecia, alopecia with brittle hair; symmetric cervical lipomas

Limb weakness may be associated with chronic progressive external ophthalmoplegia (PEO). The main features of PEO are ptosis, limitation of eye movements and diplopia. While PEO may be isolated, it is often part of KSS characterized by the association of PEO, pigment retinopathy, ataxia and heart block occurring before the age of 20 [11].

Central Nervous System A number of patients show symptoms involving predominantly or exclusively the central nervous system. These symptoms are variably associated and consist of psychomotor retardation, seizures, stroke, sensorineural hearing loss, optic atrophy, ataxia, myoclonus, peripheral neuropathy, dementia. Several clinicopathological entities such as KSS, MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), MERRF (myoclonus epilepsy and ragged red fibers), Leigh syndrome [12] and Alpers syndrome [13] have been described according to clinical

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presentations. There are several reports of patients with overlap syndromes such as KSS and MELAS, KSS and MERRF, PEO and MERRF or MERRF and MELAS. Recently, new neurological syndromes have been related to mutations in autosomalrecessive genes which affect the mRNA translation machinery and in which biochemical assessment of muscle biopsy for the mitochondrial respiratory chain enzymes is frequently normal or not helpful to address genetic confirmation. Mutations affecting elongation factors are responsible for agenesis of the corpus callosum and dysmorphism and fatal neonatal lactic acidosis [14], encephalomyopathy and hypertrophic cardiomyopathy [15], lactic acidosis, diffuse cystic leukoencephalopathy, polymicrogyria, liver involvement and early death [16]. In addition, mutations affecting mitochondrial amino-acyl-tRNA synthetase give rise to autosomal-recessive leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (aspartyl-tRNA synthetase) [17] or autosomal-recessive pontocerebellar atrophy with lactic acidosis [18].

Kidney Renal disease is more frequently reported in children than adults. The most frequent renal manifestation is proximal tubulopathy with a de Toni-Debré-Fanconi syndrome. Other renal presentations have been reported, including glomerular disease with a nephrotic syndrome and chronic tubulointerstitial nephropathy [19]. The de Toni-Debré-Fanconi syndrome is characterized by impairment of proximal tubular reabsorption leading to urinary losses of amino acids, glucose, proteins, phosphate, uric acid, bicarbonate, potassium and water. Renal Fanconi syndrome has been reported as the most frequent renal disorder in MC [19]. Proximal tubular losses are often moderate as seen in a few patients presenting hyperaminoaciduria only. Other patients present with plasma acidosis, impaired tubular phosphate reabsorption, moderate glycosuria, hypercalciuria and tubular proteinuria. The first symptoms may develop in the neonatal period and before the age of 2 years in most patients. When reported, the renal biopsy shows more or less severe unspecific anomalies of tubular epithelium with dilations or obliterations by casts, dedifferentiation or atrophy. Some tubular cells show cytoplasmic vacuolization. Giant mitochondria are frequently observed ultrastructurally. Extrarenal symptoms are generally present in all patients with mitochondrial encephalomyopathies: myopathy or other neurological symptoms, hepatic dysfunction, Pearson syndrome, cardiac involvement or diabetes mellitus, hearing loss, growth retardation. Some patients may have signs of proximal tubular acidosis with hypercalciuria. The Bartter-like phenotype has rarely been reported [20]. Glomerular disease has been observed in a few patients with MC. Patients in whom renal biopsy had shown focal and segmental glomerular sclerosis were affected by a nephrotic syndrome [19]. One of them had a decrease in the glomerular filtration rate at 9 years of age. In addition, patients had myopathy, ophthalmoplegia,

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pigmentary retinopathy, hearing loss, and hypoparathyroidism. More recently, nephrotic syndrome and segmental glomerular sclerosis has been related to disorders of Coenzyme Q10 biogenetic genes [21–23]. Renal disease in patients with mitochondrial cytopathies may also consist of tubulointerstitial nephritis. Six patients with chronic renal insufficiency without proximal tubular losses have been reported [19]. Renal biopsy in patients showed diffuse interstitial fibrosis with tubular atrophy and sclerosed glomeruli within the area of interstitial fibrosis. All patients developed extrarenal symptoms including hearing loss, cardiomyopathy, myopathy, ataxia, developmental delay, ophthalmoplegia and diabetes mellitus.

Gut Gastrointestinal symptoms include recurrent vomiting, colonic pseudo-obstruction or untreatable diarrhea. Gastrointestinal symptoms are particularly frequent in a condition called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or POLIP (polyneuropathy, ophthalmoplegia, leukoencephalopathy, and intestinal pseudoobstruction), an autosomal-recessive disease clinically defined by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities. Lossof-function mutations in thymidine phosphorylase gene induce pathologic accumulations of thymidine and deoxyuridine that in turn cause mitochondrial DNA (mtDNA) defects (depletion, multiple deletions, and point mutations). Another condition where gastrointestinal symptoms are prominent is ethylmalonic encephalopathy, a rare metabolic disorder with an autosomal-recessive mode of inheritance that is clinically characterized by neuromotor delay, hyperlactic acidemia, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea. Increased urinary levels of ethylmalonic acid and methylsuccinic acid are the main biochemical features of the disorder [24, 25]. Villous atrophy syndrome has been recognized as an mtDNA rearrangement defect. A complex III deficiency was found in the muscle of affected patients. Southern blot analysis showed evidence of heteroplasmic mtDNA rearrangements that involved deletion and deletion duplication. Two different mutations were described in the 2 children reported. In 1 patient, a deletion spanning 3,380 bp encompassed 3 genes for complex I and 3 transfer ribonuclease genes. The deletion in the second patient was larger [26].

Heart Hypertrophic cardiomyopathy is the most commonly reported type of cardiomyopathy associated with MRC disorders [27]. Cardiac hypertrophy in these disorders

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is thought to result from an increased oxidative stress, and its pathogenetic mechanism is thought to involve a cross-talk mechanism between several cell-signaling pathways as well as several transcription factors. Supraventricular tachycardia (WolfParkinson-White syndrome) have rarely been reported in association with hypertrophic cardiomyopathy. Dilated cardiomyopathy was essentially as common as hypertrophic cardiomyopathy and a change in pattern (an ‘undulating cardiomyopathy type’) can be observed [27]. Dilated cardiomyopathy is thought to result from an acute oxidative stress as a result of a high surge in reactive oxygen species, inducing damage to the mitochondrial DNA, as shown in a mouse model. Cardiac conduction disturbances have been reported in KSS with large mitochondrial DNA deletions [28]. Repeat cardiac assessment as part of monitoring disease progression is warranted in patients with confirmed OXPHOS disorders, even in the absence of clinical cardiac symptoms. Patients with OXPHOS defects who present with primary cardiac manifestations have a poorer outcome.

Endocrine System Besides common syndromes of MELAS, patients have endocrine disorders such as dysfunction with growth hormone deficiency, hypothalamopituitary hypothyroidism, hypogonadotropic hypogonadism, hypoparathyroidism, diabetes and short stature [29]. The KSS, a form of mitochondrial myopathy, is also associated with a variety of endocrine and metabolic abnormalities, in particular growth hormone deficiency (short stature), hypogonadism, diabetes mellitus, thyroid disease, hyperaldosteronism, hypomagnesemia, as well as calcification abnormalities. These patients can also show features of Pearson’s syndrome, a rare, often fatal, disorder of infancy characterized by impaired bone marrow, exocrine pancreatic, hepatic and renal function and adrenal insufficiency [30]. Finally, a few patients with diabetes mellitus have mitochondrial disorders. Patients with KSS, MELAS syndrome and Wolfram syndrome may develop diabetes mellitus [6]. The MELAS mtDNA, involving the substitution of guanine for arginine at position 3243 of tRNA leu(UUR), have been described in several families with maternally transmitted diabetes as well as in sporadic cases with insulin-dependent diabetes mellitus or with non-insulin-dependent diabetes mellitus [31]. This mutation was also reported in a family with maternally inherited cardiomyopathy, diabetes mellitus, sensorineural deafness and renal failure that was not related to diabetes mellitus [6]. It may be postulated that mutations of mtDNA in pancreatic β cells contribute to the development of diabetes mellitus.

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Liver Hepatic manifestations of mitochondrial disorders range from hepatic steatosis, cholestasis, and chronic liver disease with insidious onset to neonatal liver failure, which is frequently associated with neuromuscular symptoms multisystem involvement, and lactic acidemia. The liver disease is usually progressive and eventually fatal. Current medical therapy of mitochondrial hepatopathies is largely ineffective, and the prognosis is usually poor. These disorders include primary disorders, in which the mitochondrial defect is the primary cause of the liver disorder, and secondary disorders, in which a secondary insult to mitochondria is caused either by a genetic defect that affects non-mitochondrial proteins or by an acquired (exogenous) injury to mitochondria. Examples of secondary mitochondrial hepatopathies include Reye syndrome, Wilson’s disease, valproic acid hepatotocixity [32]. Further divided primary mitochondrial diseases into those caused by mutations affecting mtDNA genes and those caused by mutations in nuclear genes that encode mitochondrial respiratory chain proteins or cofactors. Several specific molecular defects in nuclear genes (SCO1, BCS1L, POLG, DGUOK, and MPV17 and deletion or rearrangement of mitochondrial DNA) have been identified in recent years. Four male siblings with mitochondrial cytochrome c oxidase deficiency due to compound heterozygosity for mutations in the SCO1 gene have been reported. They were affected with predominantly hepatic failure in infancy, lactic acidosis, and neurodevelopmental delays. One patient died at age 2 months and a second at the age of 5 days. Histopathologic studies of the liver showed swollen hepatocytes with microvesicular lipid vacuoles and panlobular steatosis. A muscle biopsy sample showed an accumulation of lipid droplets. SCO1 mutations are associated with COX deficiency and SCO1 is a COX assembly nuclear gene. The SCO1 gene, located at chromosome 17p13.1, is believed to encode a protein functioning as a copper chaperone that transfers copper from Cox17p, a copper-binding protein of the cytosol and mitochondrial intermembrane space, to the mitochondrial COX subunit II. A mutation in BCS1L has been found to be associated with mitochondrial neonatal liver failure. A deficient activity of complex III of the respiratory chain in the liver was reported [32], fibroblasts, or muscle in affected infants with hepatic failure, lactic acidosis, renal tubulopathy, and variable degrees of encephalopathy. Three mutations in BCS1L were demonstrated in 3 affected families. Subsequently, it was confirmed that mutations in BCS1L were associated with fatal complex III deficiency and liver failure in 2 siblings. BCS1L is a nuclear gene encoding proteins involved in the assembly of respiratory complex III [32]. The mitochondrial deoxynucleoside salvage pathway is regulated by nuclear encoded enzymes, including dGK and TK2 [32]. Human dGK phosphorylates deoxyguanosine and deoxyadenosine, whereas TK2 phosphorylates deoxythymidine,

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deoxycytidine, and deoxyuridine. An imbalance of this mitochondrial dNTP pool has been proposed to be responsible for both the hepatocerebral and myopathic forms of MC.40 In 2001, mutations in 2 genes involved in this pathway were identified in patients with MC:deoxyguanosine kinase (DGUOK) in the hepatocerebral form and TK2 in the myopathic form [32]. Mandel et al. [33] using homozygosity mapping in 3 consanguineous kindreds affected with hepatocerebral MC, mapped this disease to chromosome 2p13, which encompasses the gene DGUOK encoding dGK. A single-nucleotide deletion (204delA) within the coding region of DGUOK was identified. The reduction of enzymatic activities of mitochondrial respiratory chain complexes containing mtDNA encoded subunits (complexes I, III and IV but not complex II, which is solely encoded by nuclear genes) was demonstrated in the liver but not in muscle, showing the tissuespecific nature of this disorder. However, Salviati et al. [34] screened the frequency of DGUOK mutations in 21 patients with hepatocerebral MC and noted that DGUOK mutations were present in only 14%, suggesting this was not the only gene responsible for MC in the liver. Patients with DGUOK mutations present with lactic acidosis, hepatomegaly, hypoglycemia, jaundice, and encephalopathy with hypotonia, hyperreflexia, and nystagmus, and oculogyric crises. Two other nuclear genes have recently been linked to the hepatocerebral form of MC [9, 35], POLG and MPV17. Mutations in DNA POLG, which is confined to mitochondria but encoded by a nuclear gene, have now been described in infants with MC and in older children with Alpers-Huttenlocher disease. Most of the cases with MC in early childhood are associated with at least 1 mutation in the linker region of POLG and 1 in the polymerase domain. More recently, Spinazzola et al. [35] used a novel integrative genomics approach to discover mutations in the nuclear gene MPV17 in 3 families affected by the hepatocerebral form of MC. This gene encodes an inner mitochondrial membrane protein of uncertain function. Mutations in POLG have recently been shown to be common in patients with Alpers-Huttenlocher syndrome. The mtDNA polymerase g (POLG) is essential for mtDNA replication and repair. Pol g is composed of a 140-kDa catalytic (α) subunit that contains DNA polymerase, 3–5 exonuclease, and dRP (deoxyribose phosphate) lyase activity and a 55-kDa accessory (β) subunit that functions as a progressive and DNA-binding factor. In Alpers-Huttenlocher syndrome, children are usually normal at birth with some developmental delay in infancy and often present with hypotonia and bouts of vomiting. The seizure disorder usually has an abrupt onset and although clinical signs of liver disease often appear later, biochemical evidence of liver disease is sometimes present before the onset of seizures. EEG and visual-evoked potentials are abnormal. Most patients die before the age of 3 years. Less frequently, late presentation occurs, even up to 25 years of age. Some patients also have visual disturbances. Pathologic liver findings, including fatty changes, abnormal bile duct architecture and fibrosis,

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are unrelated to anticonvulsant therapy. Neuropathology shows severe cortical neurodegeneration and astrocytosis. A deficiency in mitochondrial POLG activity and mtDNA depletion was first reported in a patient with Alpers-Huttenlocher syndrome in 1999 [9]. Naviaux and Nguyen [9] reported that in 2 unrelated pedigrees with Alpers-Huttenlocher syndrome, each affected child was found to harbor a homozygous mutations in exon 17 of POLG that led to a Glu873Stop mutation just upstream of the polymerase domain of the protein. In addition, each affected child was heterozygous for the G1681A mutation in exon 7, which led to an Ala467Thr substitution in POLG, within the linker region of the protein. Vu et al. [36] first demonstrated mtDNA depletion in liver biopsies from 2 patients with NNH (Navajo neurohepatopathy), which was consistent with the hypothesis that a nuclear gene might be responsible for this autosomal-recessive disease. Navajo neurohepatopathy is an autosomal-recessive multisystem disorder prevalent in the Navajo population of the southwestern United States. Patients with NNH present with liver disease, severe sensory and motor neuropathy, corneal anesthesia and scarring, cerebral leukoencephalopathy, failure to thrive, and recurrent metabolic acidosis with intercurrent illness. A genomewide scan, performed with 400 DNA microsatellite markers, demonstrated mapping of the disease to chromosome 2p24.1. The gene was MPV17 [37], involved in mtDNA maintenance and in the regulation of OXPHOS and is localized to the inner mitochondrial membrane. The sequencing of MPV17 in 6 NNH patients from 5 families in 2006 demonstrated the same homozygous diseasecausing R50Q mutations in exon 2 in all patients, confirming a founder effect in this disease. Thus, it is now clear that NNH is indeed a form of mtDNA depletion with a unique clinical presentation in Navajos. Prospective, longitudinal multicenter studies will be needed to address the gaps in our knowledge in these rare liver diseases. The role of liver transplantation in patients with liver failure remains poorly defined because of the systemic nature of the disease that does not respond to transplantation.

Blood Hematological manifestations of mitochondrial cytopathies include aplastic, megaloblastic or sideroblastic anemia, leukopenia, neutropenia, thrombocytopenia, or pancytopenia. In single cases, either permanent or recurrent eosinophilia has been observed. Hematological abnormalities may occur together with syndromic or nonsyndromic mitochondrial cytopathies. Syndromic mitochondrial cytopathies in which hematological manifestations predominate are Pearson syndrome (pancytopenia), KSS (anemia), Barth syndrome (neutropenia), and autosomal-recessive mitochondrial myopathy, lactic acidosis and sideroblastic anemia syndrome (MLASA) [38]. The association of Pearson syndrome and a specific deletion in mtDNA was

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first reported in 1990 by Rotig’s group. It is now established that mtDNA rearrangements are present in all patients with Pearson syndrome, with large (4,000–5,000 bp) deletions predominating in three quarters of the reported cases. The most common deletion is located between nt 8488 and nt 13,460. The proteins affected by this deletion include respiratory chain enzymes (complex I is the most severely affected), 2 subunits of complex V, 1 subunit of complex IV, and 5 transfer RNA genes. Other mtDNA deletions of differing lengths are associated with clusters of the characteristic clinical manifestations. Another rare condition of mitochondrial cytopathy is X-linked sideroblastic anemia with ataxia (XLSA/A), a rare syndromic form of inherited sideroblastic anemia associated with spinocerebellar ataxia, due to mutations in the mitochondrial ATPbinding cassette transporter Abcb7 [39]. Anemia has been described in single cases with Leigh syndrome, MERRF (myoclonic epilepsy and ragged-red fiber) syndrome, or Leber’s hereditary optic neuropathy. Anemia, leukopenia, thrombocytopenia, eosinophilia, or pancytopenia can also be found frequently in nonsyndromic mitochondrial cytopathies with or without involvement of other tissues. Therapy of blood cell involvement in MID comprises application of antioxidants, vitamins, iron, bone marrow-stimulating factors, or substitution of cells.

Skin Reviewing the literature of skin disorders associated with mitochondrial encephalomyopathies, the most recurring sign is lipomas. Symmetric cervical lipomas are a presenting feature of Ekbom’s syndrome of cervical lipomas associated with myoclonic epilepsy and ragged red muscle fibers (MERRF) and mutations in mtDNA tRNA LysUUR 8344A-G [40]. Excluding lipomas, skin findings such as discoloration, hirsutism, and anhidrosis have been reported in several other patients. Pigment alterations consistent with poikiloderma, i.e. acrocyanosis and vitiligo, have been reported in some patients. Acrocyanosis has been reported together with Pearson syndrome, and the presence of episodic acrocyanosis is part of the syndromic association of ethylmalonic encephalopathy (EE), a devastating infantile metabolic disorder affecting the brain, gastrointestinal tract, and peripheral vessels. The gene responsible for EE is ETHE1 [24] and the principal signs of the syndrome are recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea. Other discolorations of the skin have been reported such as periorbital darkening, generalized hyperpigmentation associated with adrenal insufficiency, cutis marmorata and jaundice in patients with the hepatocerebral form of mitochondrial DNA depletion syndrome (MDDS) due to mutations in the nuclear-encoded mitochondrial deoxyguanosine kinase gene DGUOK [33] or in the MPV17 gene [35].

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Another characteristic finding of mitochondrial encephalomyopathies is hirsutism or hypertrichosis that has been reported in patients with Pearson syndrome and quite frequently in severe Leigh syndrome with encephalomayopathy, citochome-coxidase deficiency and mutations in the SURF-1 gene. In addition alopecia, alopecia with brittle hair, or trichothiodystrophy have also been reported [40].

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18 Edvardson S, Shaag A, Kolesnikova O, Gomori JM, Tarassov I, Einbinder T, Saada A, Elpeleg O: Deleterious mutation in the mitochondrial arginyltransfer RNA synthetase gene is associated with pontocerebellar hypoplasia. Am J Hum Genet. 2007; 81:857–862. 19 Nialdet P, Rotig A: The kidney in mitochondrial cytopathies. Kidney Int 1997;51:1000–1007. 20 Emma F, Pizzini C, Tessa A, Di Giandomenico S, Onetti-Muda A, Santorelli FM, Bertini E, Rizzoni G: ‘Bartter-like’ phenotype in Kearns-Sayre syndrome. Pediatr Nephrol 2006;21:355–360. 21 DiMauro S, Quinzii CM, Hirano M. Mutations in coenzyme Q10 biosynthetic genes. J Clin Invest. 2007;117:587–589. 22 López LC, Schuelke M, Quinzii CM, Kanki T, Rodenburg RJ, Naini A, Dimauro S, Hirano M: Leigh syndrome with nephropathy and CoQ10 deficiency due to decaprenyl diphosphate synthase subunit 2 (PDSS2) mutations. Am J Hum Genet 2006; 79:1125–1129. 23 Diomedi-Camassei F, Di Giandomenico S, Santorelli FM, Caridi G, Piemonte F, Montini G, Ghiggeri GM, Murer L, Barisoni L, Pastore A, Muda AO, Valente ML, Bertini E, Emma F: COQ2 nephropathy: a newly described inherited mitochondriopathy with primary renal involvement. J Am Soc Nephrol 2007;18:2773–2780. 24 Tiranti V, Briem E, Lamantea E, Mineri R, Papaleo E, De Gioia L, Forlani F, Rinaldo P, Dickson P, AbuLibdeh B, Cindro-Heberle L, Owaidha M, Jack RM, Christensen E, Burlina A, Zeviani M: ETHE1 mutations are specific to ethylmalonic encephalopathy. J Med Genet 2006;43:340–346. 25 Hirano M, Nishino I, Nishigaki Y, Martí R: Thymidine phosphorylase gene mutations cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Intern Med 2006;45:1103. 26 Cormier-Daire V, Bonnefont JP, Rustin P, Maurage C, Ogier H, Schmitz J, Ricour C, Saudubray JM, Munnich A, Rötig A: Mitochondrial DNA rearrangement with onset as chronic diarrhea with villous atrophy. J Pediatr 1994;124:63–70. 27 Yaplito-Lee J, Weintraub R,. Jamsen K, Chow C W, ThorburnD R, Boneh A: Cardiac manifestations in oxidative phosphorylation disorders of childhood. J Pediatr 2007;150:407–411. 28 Anan R, Nakagawa M, Miyata M, Higuchi I, Nakao S, Suehara M, Osame M, Tanaka H: Cardiac involvement in mitochondrial diseases: a study on 17 patients with documented mitochondrial DNA defects. Circulation 1995;91:955–961. 29 Stark R and Roden M. Mitochondrial function and endocrine diseases. Eur J Clin Invest 2007;37:236– 248.

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30 Bruno C, Minetti C, Tang Y, Magalhães PJ, Santorelli FM, Shanske S, Bado M, Cordone G, Gatti R, DiMauro S: Primary adrenal insufficiency in a child with a mitochondrial DNA deletion. J Inherit Metab Dis 1998;21:155–161. 31 Kadowaki T, Kadowaki H, Mori Y, Tobe K, Sakuta R, Suzuki Y, Tanabe Y, Sakura H, Awata T, Goto Y, Hayakawa T, Matsuoka K, Kamada RKT, Horai S, Ikuya Nonaka I, Hagura R, Akanuma Y, Yazaki Y: A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N Engl J Med 1994;330: 962–968. 32 Lee WS, Sokol RJ: Mitochondrial hepatopathies: advances in genetics and pathogenesis. Hepatology 2007;45:1555–1565. 33 Mandel H, Szargel R, Labay V, Elpeleg O, Saada A, Shalata A, Anbinder Y, Berkowitz D, Hartman C, Barak M, Eriksson S, Cohen N: The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. Nat Genet 2001;29:337–340. 34 Salviati L, Sacconi S, Mancuso M, Otaegui D, Camaño P, Marina A, Rabinowitz S, Shiffman R, Thompson K, Wilson CM, Feigenbaum A, Naini AB, Hirano M, Bonilla E, DiMauro S, Vu TH: Mitochondrial DNA depletion and dGK gene mutations. Ann Neurol 2002;52:311–317. 35 Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D’Adamo P, Calvo S, Marsano RM, Donnini C, Weiher H, Strisciuglio P, Parini R, Sarzi E, Chan A, DiMauro S, Rötig A, Gasparini P, Ferrero I, Mootha VK, Tiranti V, Zeviani M: MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat Genet 2006;38:570–575. 36 Vu TH, Tanji K, Holve SA, Bonilla E, Sokol RJ, Snyder RD, Fiore S, Deutsch GH, Dimauro S, De Vivo D: Navajo neurohepatopathy: a mitochondrial DNA depletion syndrome? Hepatology 2001;34:116– 120. 37 Karadimas CL, Vu TH, Holve SA, Chronopoulou P, Quinzii C, Johnsen SD, Kurth J, Eggers E, Palenzuela L, Tanji K, Bonilla E, De Vivo DC, DiMauro S, Hirano M. Navajo neurohepatopathy is caused by a mutation in the MPV17 gene. Am J Hum Genet 2006;79:544–548. 38 Fernandez-Vizarra E, Berardinelli A, Valente L, Tiranti V, Zeviani M: Nonsense mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by myopathy, lactic acidosis and sideroblastic anaemia (MLASA). J Med Genet 2007;44:173– 180.

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Enrico Bertini, MD Bambino Gesu’ Children’s Research Hospital Unit of Molecular Medicine for Neuromuscular and Neurodegenerative Disorders Department of Laboratory Medicine, IT–00165 Rome (Italy) Tel. +39 06 6859 2105, Fax +39 06 6859 2024, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 53–60

Overgrowth Syndromes: A Classification Giovanni Neri ⭈ Marco Moscarda Institute of Medical Genetics, Catholic University, Rome, Italy

Abstract Overgrowth syndromes are characterized by macrosomia, congenital anomalies, mental retardation and an increased risk of tumors. In this article we will analyze what we define ‘classical’ overgrowth syndromes (Beckwith-Wiedemann, Simpson-Golabi-Behmel, Sotos, Weaver and Perlman syndromes) and will trace a brief overview of other conditions with excessive growth. Copyright © 2009 S. Karger AG, Basel

Congenital ‘overgrowth’ is defined by a neonatal weight above the 97th centile, while in childhood and adulthood overgrowth is defined by height (above the 97th centile) rather than by weight. Secondary causes of high birth weight, like maternal diabetes mellitus or obesity, should be ruled out before considering the diagnosis of constitutional infant macrosomia. Due to the frequently overlapping findings, a diagnostic strategy may be in order, as proposed by Boccuto et al. [1]. Familial history and some specific clinical characteristics may be relevant in selecting molecular tests to confirm the diagnosis. In particular, we shall describe conditions with overgrowth as the main finding of the clinical presentation, like Beckwith-Wiedemann, SimpsonGolabi-Behmel, Sotos, Weaver and Perlman syndromes. We shall also describe some other conditions that have macrosomia as an accessory finding and for which a laboratory test is available. These are the Bannayan-Riley-Ruvalcaba, the Pallister Killian, and the 22q13 deletion syndromes.

Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS) (OMIM #130650) is the most common overgrowth syndrome with an estimated incidence of 1/13,700 liveborns. Major clinical findings are omphalocele, macrosomia and macroglossia that can be accompanied by other manifestations, delineating a broader phenotypic spectrum.

Table 1. Relative frequency and associated recurrence and tumor risk for molecular defects involved in BWS Defect (frequency)

Recurrence in sibs

Tumor risk

Loss of methylation of LIT1 (40–50%)

sporadic

lower

Paternal UPD (20%)

sporadic

higher

CDKN1C mutation (5%, 40% of familial BWS)

up to 50%

lower

Gain of methylation of H19 (5–10%)

sporadic

higher

Paternal duplication or maternal rearrangement of 11p15.5 (2%)

up to 50% if parent is a carrier

IC1 or IC2 deletion (1%) Unknown defect (15–25%)

Hemihyperplasia (asymmetric overgrowth of one or more regions of the body) occurs in about 25% of cases and is thought to result from somatic mosaicism. In most cases, hemihyperplasia is not manifest at birth. Adult height is generally normal, indicating a deceleration of the initial excess of growth. The face may be coarse with prominent metopic suture, large anterior fontanelle, prominent occiput, midfacial hypoplasia and infraorbital creases. Other important characteristics can be the presence of anterior ear lobe creases/posterior helical ear pits, cleft palate, visceromegaly (i.e. hepatomegaly, also detectable prenatally), renal abnormalities. Polyhydramnios and prematurity, neonatal hypoglycemia, facial nevus flammeus, capillary dysplasias, cardiac anomalies (9–34% of patients), diastasis recti, advanced bone age (especially in the first 4 years of life), monozygotic twinning are less frequent findings. There is a 20% incidence of perinatal death due to complications of prematurity, macroglossia or, rarely, cardiomyopathy. IQ is generally normal, unless there is a concomitant chromosomal imbalance extending beyond the 11p15 region, which contains the genes responsible for BWS. For a comprehensive review of diagnostic criteria, see Weksberg et al. [2]. BWS constitutes a risk factor for developing infantile malignancies (7.5–12.5%). The most common occurrences are Wilms’ tumor, hepatoblastoma, and neuroblastoma, although rhabdomyosarcoma and many other malignant and benign tumors have also been reported. Hemihyperplasia confers an increased cancer risk, even on the side not affected by overgrowth. Nephromegaly and nephrogenic rests or nephroblastomatosis are considered precursors of Wilms’ tumor. After mid-childhood (age 8), BWS patients are no longer considered to be at increased risk of malignancies. BWS is caused by altered expression of growth regulatory genes on the imprinted regions of chromosome 11p15. IGF2 (insulin-like growth factor 2) and LIT1 are expressed only in the paternal allele and promote cell growth. On the other hand, H19 and CDKN1C, which negatively regulate cell proliferation, and KvLQT1, are expressed

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in the maternal allele. These genes are controlled by 2 different imprinting centers: the telomeric IC1, regulating H19 and IGF2, and the centromeric IC2, controlling LIT1 and CDKN1C. Gene dosage imbalance leading to BWS may be caused by chromosomal rearrangements, paternal uniparental disomy, loss of methylation in the maternal allele of LIT1, gain of methylation in the maternal allele of H19, mutations in CDKN1C, deletions of the IC1 or IC2. For a review about molecular mechanisms involved in BWS, see Cytrynbaum et al. [3]. Defects involving IC1 are associated with higher tumor risk, in particular Wilms tumor, while IC2 alterations are correlated with a lower cancer risk (table 1). A diagnosis of BWS in infancy should follow up with a detailed tumor surveillance protocol, including abdominal ultrasound screening every three months until age 8 years [4]. Assisted reproductive technology (ART) has been investigated as a possible risk factor for BWS. However, a molecular mechanism still has to be defined and further studies are needed [5].

Simpson-Golabi-Behmel Syndrome

Simpson-Golabi-Behmel syndrome (SGBS) (OMIM #312870) is an X-linked semidominant condition characterized by a wide phenotypic spectrum, from mildly affected female carriers to severely affected males, with a high risk of neonatal death. The main clinical findings are pre- and postnatal overgrowth, congenital hypotonia and multiple malformations, including heart defects, polydactyly, supernumerary nipples [6]. Prenatal overgrowth in affected males persists postnatally, with final height usually above the 97th centile. The face is typical, with hypertelorism, downslanting palpebral fissures, epicanthic folds, low-set, posteriorly angulated ears, macrostomia with macroglossia and dental malocclusion, midline groove of lower lip, short nose with anteverted nares, short or webbed neck. Other common findings are macrocephaly, rib and vertebral anomalies, advanced bone age, hepatosplenomegaly, neonatal hypoglycemia. Hands and feet are usually broad with polydactyly, cutaneous syndactyly, clino-/camptodactyly, and fingernail hypoplasia, especially of the 2nd finger. Multicystic dysplastic kidneys, hyperplastic islets of Langherans, cryptorchidism, diaphragmatic defects, diastasis recti, umbilical and inguinal hernias and early death are less common. SGBS has an increased risk of embryonal and childhood tumors, like Wilms’ tumor and neuroblastoma, but also hepatocellular carcinoma and testicular gonadoblastoma. The syndrome is caused by mutations in the GPC3 gene, mapped to Xq26 [7]. GPC3 is probably a negative regulator of cell proliferation and apoptosis, through modulation of cellular responses to growth factors, possibly involving the IGF-II pathway. The gene is highly expressed during fetal development, especially in those tissues that are most affected by overgrowth. The genetic test is indicated in all cases with a clinical diagnosis of SGBS, although a confirmatory results is usually seen in no more than about half of the tested cases.

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Sotos Syndrome

Sotos syndrome (SoS) (OMIM #117550) is characterized by overgrowth of prenatal onset persisting during the first years of life, advanced bone age and characteristic facial features [8]. Birth weight averages 4,200 g in males and 4,000 g in females, while mean birth length is 55.6 cm in males and 57.3 cm in females Occipitofrontal head circumference (OFC) tends to exceed the 97th centile by the age of 1 year. However, final height is usually within normal range, due to the advanced bone age, present in 84% of SoS patients. Advanced bone age is common to many overgrowth syndromes, including Weaver, BWS and SGBS. However, SoS can be distinguished for the presence of a peculiar (disharmonic) metacarpophalangeal profile. Hand and foot length is often above the 97th centile; dental eruption can occur early; puberty can be precocious. Facial characteristics are marked frontal bossing (in 97.5% of patients), high frontal hairline (97.5%), frontoparietal balding, downslanting palpebral fissures (90%), narrow bitemporal diameter, full cheeks, high palate (70%). The face gradually lengthens with age and the jaw becomes more prominent, with a pointed chin. Congenital hypotonia and poor feeding affect the neonatal period, while in childhood there is delay in expressive language. IQ is borderline (mean of 78, ranging from 40 to 129). Autistic behavior may also be noted. Pes planus is frequent, strabismus is found in nearly 40% of patients, and seizures in 50%. MRI can show a typical pattern characterized by prominence of the trigonus (90%) and of the occipital horns (75%) and ventriculomegaly (63%). The supratentorial extracerebral fluid spaces and those of the posterior fossa are increased for age in 70% of patients. Heart defects (i.e. septal defects and patent ductus arteriosus) are observed in 8% of SoS patients, but seem to be as frequent as 35–41% in patients of Japanese ethnicity. Gastrointestinal and urogenital anomalies have also been reported. Upper respiratory tract infections, especially otitis media, are frequent and may lead to conductive hearing loss. Cohen [9] critically reviewed neoplasms reported to occur with SoS. One third of patients with neoplasia, all males, developed lympho-hematological malignancies (lymphoma or leukemia), which seem to represent the most frequent neoplasias in SoS. Many cases occurred after the age of 5 (10/22; 45%) or 10 years (4/22; 18%). Thus, it is not easy to give an overall recommendation for cancer surveillance in SoS. The NSD1 gene (nuclear-receptor- binding SET-domain-containing protein 1) was first identified as causing agent of SoS in patients with a chromosomal translocation, encompassing chromosomal band 5q35. Haploinsufficiency of NSD1 has been confirmed as the major cause of SoS [10]. A recurrent 2.2-Mb microdeletion at 5q35, including NSD1 and adjacent genes, is the primary cause of SoS in Japan, accounting for over 50% of cases. In Europe and USA intragenic mutations cause 60–80% of SoS cases, whereas microdeletions are responsible for no more than 10% of cases. Partial NSD1 deletions, encompassing one or more exons, were detected in SoS cases without NSD1 mutation or 5q35 microdeletion [11].

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Weaver Syndrome

Weaver syndrome (WS) (OMIM #277590) is characterized by persistent overgrowth of prenatal onset, accelerated bone maturation, distinctive craniofacial appearance, developmental delay, a hoarse and low-pitched cry, congenital hypotonia, widened distal long bones and camptodactyly. Mean birth weight is 4,785 g in males and 3,883 g in females, length 56 and 53 cm, and OFC 36.6 and 35.2 cm, respectively. Average final height is 194.2 cm in males and 176.3 cm in females, with an average weight of 102.2 kg in males and 87.6 kg in females, OFC averages 61 cm in males and 59.5 cm in females. Skeletal growth is more accelerated than skeletal maturation, resulting in excessive height in adults. There may also be large low-set ears, a large bifrontal diameter, flat occiput, hypertelorism, long upper lip with prominent philtrum, micrognathia, postnatal persistence of fingerpads, clinodactyly of fifth finger, broad thumbs, transverse palmar crease(s), thin hyperconvex and deep-set nails, and finger camptodactyly [12]. Limited extension at elbows and knees may be secondary to reduced prenatal movements. Clubfoot may be unilateral or bilateral. Brain MRI findings include cavum septi pellucidi and dilated ventricles. Skeletal X-rays may show short fourth metatarsals, wide distal metaphyses of femora (and somewhat mottled epiphyses), and low, broad iliac wings. Loose skin and inverted nipples may result from resorption of congenital lymphedema. Hair may be sparse. Umbilical and inguinal hernias are common. Mild hypertonicity or hypotonia are common, with mild-to-moderate psychomotor delay that improves with age, so that IQ is usually normal or ‘borderline’. Young children may have breathing and swallowing difficulties. Douglas et al. [13] analyzed 7 WS patients and identified a NSD1 mutation in 3 (42%). However, these WS cases were later re-diagnosed as SoS. Therefore, the cause of WS remains unidentified.

Perlman Syndrome

Perlman syndrome (PS) (OMIM #267000) is a rare, autosomal-recessive condition characterized by nephromegaly, macrosomia, hypotonia, cryptorchidism and a typical facial appearance, with a full round face, prominent forehead, deep-set eyes with epicanthic folds, broad depressed nasal bridge, everted upper lip, micrognathia and highly arched palate [14]. The chest may be broad with pectus excavatum, the abdomen distended due to visceromegaly. Hyperplasia of the islets of Langerhans is frequent and can be related to congenital macrosomia and hypoglycaemia, leading to infant death. Sex ratio is reported as 2 M:1 F. There may be a history of polyhydramnios and fetal ascites. PS is a severe condition and most children die neonatally. In the few survivors growth pattern quickly falls within or below normal range. Wilms’ tumor is the only neoplasia whose risk is increased in PS, due to the high prevalence of nephroblastomatosis and nephrogenic rests. Autosomal-recessive inheritance has

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been assumed since the observation of multiple affected siblings in consanguineous as well as nonconsanguineous families.

Bannayan-Riley-Ruvalcaba Syndrome

Bannayan-Riley-Ruvalcaba syndrome (BRRS) (OMIM #153480) is an autosomaldominant disorder consisting of macrocephaly, vascular malformations, lipomas (75% of patients), hamartomatous polyps of the distal ileum and colon (45% of patients), pigmented macules on the shaft of the penis and Hashimoto thyroiditis [15]. Birth weight usually exceeds 4,000 g and birth length is above the 97th centile. Postnatal growth decelerates, so older children and adults fall within the normal height range. Head circumference is at least 4.5 SD above the mean. Hypotonia, gross motor delay, mild-to-severe mental retardation and cognitive speech delay may be present in approximately 70% of patients, while seizures in about 25%. Asymmetric motor development has been reported, as well as a lipid storage myopathy involving neutral fat accumulation, predominantly in enlarged type I muscle fibers, and reduced muscle-free carnitine levels. This myopathic process affects the proximal muscles in at least 60% of BRRS patients. Pigmented macules on the penile shaft are a peculiar finding of the syndrome and may develop in late childhood. Ocular abnormalities have occasionally been reported, such as hypertelorism, downslanting palpebral fissures, strabismus, amblyopia, prominent Schwalbe lines, visible corneal nerves and pseudopapilledema. Joint hyperextensibility, pectus excavatum, scoliosis have been recorded in about 50% of patients. Facial acanthosis-like lesions and accessory nipples may also be present. BRRS is caused by mutations in the PTEN gene (phosphatase and tensin homologue deleted on chromosome 10). PTEN is considered a tumor suppressor gene with an important role in two critical pathways for proliferation and differentiation: the PI3/AKT and the mitogen-activated kinase (MAPK) pathways. PTEN haploinsufficiency was detected in the wide majority of cases of both familial and sporadic BRRS and was found to be correlated with the presence of breast tumors, breast fibroadenomas and lipomatosis [16].

22q13 Deletion Syndrome

The 22q13 deletion syndrome (OMIM #606232) is characterized by normal or accelerated growth in 95% of patients, hypotonia in 97%, absent or delayed speech, global developmental delay and minor anomalies, including dolichocephaly, abnormal ears, pointed chin, palpebral ptosis, epicanthic folds, saddle nose with bulbous tip, fleshy hands, dysplastic toenails, increased tolerance to pain, stereotypic movements (chewing) and tendency to overheating due to poor perspiration [17]. Autism and other behavioral disorders have been frequently described in these patients. Differential

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diagnosis of a case with generalized hypotonia in a newborn with no recognizable cause should take into consideration either the del 22q13 syndrome or the PraderWilli syndrome. In older infants and children, overgrowth, hypotonia and developmental delay, associated with absent or severely delayed speech, autistic-like behavior, and minor craniofacial anomalies, should suggest del 22q13 as a possible diagnosis. The 22q13 deletion interval includes the SHANK3 gene, whose haploinsufficiency is responsible for at least some cases of autism. [18]. Interestingly, autism in males is often accompanied by macrocephaly.

Pallister-Killian Syndrome

Pallister-Killian syndrome (PKS) (OMIM #60183) is a rare, sporadic multiple congenital anomaly/mental retardation syndrome, caused by tissue-limited mosaicism of a supernumerary 12p isochromosome [i(12)(p10)]. Typical clinical findings are abnormal pigmentary streaks of the skin, mental retardation, congenital hypotonia, seizures and facial anomalies, such as hypertelorism, epicanthal folds, cataracts, sparse eyebrows and eyelashes, abnormal ears, macrostomia with ‘cupid-bow’ shape of upper lip and pouting lower lip, a broad high forehead, short nose with anteverted nostrils, broad nasal bridge, full cheeks, sparseness of scalp hair, short neck. Hearing deficit, congenital heart defects, accessory nipples, distal digital hypoplasia, diaphragmatic hernia, anal and genito-urinary abnormalities are also present [19]. Growth pattern is characterized by normal to increased birth length with postnatal deceleration of length, normal to increased birth weight with postnatal obesity, and normal to increased OFC at birth, with postnatal deceleration of its growth. In PKS, the average birth weight adjusted for term is about 3,600 g, with individual patients varying from the 75th to the 90th centile. PKS doesn’t seem to be associated with increased risk of neoplasia. Prenatal diagnosis of PKS can be performed by chorionic villus sampling (CVS) from the end of the first trimester of pregnancy, or by amniocentesis or fetal blood sampling. In all cases, FISH analysis is indicated to confirm the diagnosis. In PKS patients, peripheral blood lymphocytes usually show normal chromosomes; isochromosome 12p may be found in skin fibroblasts or bone marrow cells. Zollino et al. [20] reported mitotic instability of the i(12p) clones that may may create a problem with respect to prenatal cytogenetic diagnosis.

References 1

2

Boccuto L, Lapunzina P, Gurrieri F, Neri G. Diagnostic strategies in overgrowth syndromes. Ital J Pediatr 2006;32:81–100. Weksberg R, Shuman C, Smith A: BeckwithWiedemann syndrome. Am J Hum Gen 2005;137C: 12–23.

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Cytrynbaum CS, Smith AC, Rubin T, Weksberg R: Advances in overgrowth syndromes: clinical classification to molecular delineation in Sotos syndrome and Beckwith-Wiedemann syndrome. Curr Opin Pediatr 2005;17:740–706.

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4 Rao A, Rothman J, Nichols KE: Genetic testing and tumor surveillance for children with cancer predisposition syndromes. Curr Opin Pediatr 2008;20: 1–7. 5 Lawrence LT, Moley KH: Epigenetics and assisted reproductive technologies: human imprinting syndromes. Semin Reprod Med. 2008;26:143–152. 6 Neri G, Gurrieri F, Zanni G, Lin A: Clinical and molecular aspects of the Simpson-Golabi-Behmel syndrome. Am J Med Genet 1998;79:279–283. 7 DeBaun MR, Ess J, Saunders S: Simpson Golabi Behmel syndrome: progress toward understanding the molecular basis for overgrowth, malformation, and cancer predisposition. Mol Genet Metab 2001; 72:279–286. 8 Baujat G, Cormier-Daire V: Sotos syndrome. Orphanet J Rare Dis 2007;2:36. 9 Cohen MM Jr: Mental deficiency, alterations in performance, and CNS abnormalities in overgrowth syndromes. Am J Med Genet [C] 2003;117:49–56. 10 Niikawa N: Molecular basis of Sotos syndrome. Horm Res 2004;62(suppl 3):60–65. 11 Douglas J, Tatton-Brown K, Coleman K, Guerrero S, Berg J, Cole TR, Fitzpatrick D, Gillerot Y, Hughes HE, Pilz D, Raymond FL, Temple IK, Irrthum A, Schouten JP, Rahman N: Partial NSD1 deletions cause 5% of Sotos syndrome and are readily identifiable by multiplex ligation dependent probe amplification. J Med Genet 2005;42:e5. 12 Opitz JM, Weaver DW, Reynolds JF Jr: The syndromes of Sotos and Weaver: reports and review. Am J Med Genet 1998;79:294–304. 13 Douglas J, Hanks S, Temple IK, Davies S, Murray A, Upadhyaya M, Tomkins S, Hughes HE, Cole TR, Rahman N: NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes. Am J Hum Genet 2003;72:132–143.

14 Henneveld HT, van Lingen RA, Hamel BC, StolteDijkstra I, van Essen AJ: Perlman syndrome: four additional cases and review. Am J Med Genet 1999; 86:439–446. 15 Nowak CB: The phakomatoses: dermatologic clues to neurologic anomalies. Semin Pediatr Neurol 2007;14:140–149. 16 Marsh DJ, Kum JB, Lunetta KL, et al: PTEN mutation spectrum and genotype-phenotype correlations in Bannayan-Riley-Ruvalcaba syndrome suggest a single entity with Cowden syndrome. Hum Mol Genet 1999;8:1461–1472. 17 Cusmano-Ozog K, Manning MA, Hoyme HE: 22q13.3 deletion syndrome: a recognizable malformation syndrome associated with marked speech and language delay. Am J Med Genet [C] 2007;145: 393–398. 18 Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, Nygren G, Rastam M, Gillberg IC, Anckarsäter H, Sponheim E, GoubranBotros H, Delorme R, Chabane N, Mouren-Simeoni MC, de Mas P, Bieth E, Rogé B, Héron D, Burglen L, Gillberg C, Leboyer M, Bourgeron T: Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet 2007;39:25–27. 19 Bielanska MM, Khalifa MM, Duncan AM: PallisterKillian syndrome: a mild case diagnosed by fluorescence in situ hybridization: review of the literature and expansion of the phenotype. Am J Med Genet 1996;65:104–108. 20 Zollino M, Bajer J, Neri G: Chromosome instability limited to the aneuploid clone in the Pallister-Killian syndrome: a pitfall in prenatal diagnosis. Prenat Diagn 1999;19:184–185.

Giovanni Neri, MD Istituto di Genetica Medica, Università Cattolica Largo F. Vito, 1 IT–168 Roma (Italy) Tel. +39 06 305 4449, Fax +39 06 305 0031, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 61–66

C-Type Natriuretic Peptide and Overgrowth Renata Bocciardia Roberto Ravazzoloa,b a Laboratory of Molecular Genetics, G. Gaslini Institute, and bDepartment of Pediatrics, University of Genova and CEBR, Genova, Italy

Abstract Natriuretic peptides are a family of structurally related peptides with different distinct biological effects. C-type natriuretic peptide (CNP)-mediated signaling is important for endochondral ossification and intervenes in the control of chondrocyte maturation by regulating the balance between proliferation and terminal differentiation. CNP is encoded by the NPPC gene on human chromosome 2 for which, so far, no mutations have been described in humans. Recently, two independent articles reported the description of 3 patients with a similar clinical phenotype characterized by the presence of skeletal anomalies and overgrowth. In all 3 cases, the clinical picture was associated with the presence of a balanced translocation involving chromosome 2 and causing overexpression of the NPPC gene and an increased plasma concentration of its product, CNP. Transcriptional dysregulation of NPPC has been ascribed to the separation of the gene unit from the long-range regulatory element with a transcriptional silencing effect on its expression and CNP overproduction has been corCopyright © 2009 S. Karger AG, Basel related to the skeletal overgrowth phenotype observed.

The formation of a skeleton occurs through two different mechanisms: endochondral and membranous ossification. Endochondral ossification involves sequential steps beginning from the shaping of a cartilaginous anlage and culminating in the formation of bones. Briefly, the proliferating mesenchymal cells condense and start to differentiate into chondrocytes. Commitment to the skeletal lineage progresses through a switch in gene activation and results in the production of a cartilage matrix. As development proceeds, chondrocytes enter a process of maturation, characterized by cellular hypertrophy and the onset of type X collagen expression. Finally, chondrocytes die and, concomitant with blood invasion, cartilage is gradually replaced by bone [1]. This process is responsible for longitudinal bone growth, both during embryonic development and in postnatal life. In this latter case, it occurs at the cartilaginous growth plates located at both ends of the vertebrae and long bones. Chondrocyte maturation must be precisely controlled, with a finely tuned balance between proliferation

Nppc/ mice Increased mortality, jaw malocclusion, small rib cage causing pulmonary restriction Dwarfism due to shortening of long bones and vertebrae. Transgenic mice overexpressing Nppc Skeletal overgrowth; expansion of both proliferative and hypertrophic regions of the long bones growth plates

Targeted deletion of selected domains of NPR-B Dwarfism and female infertility Spontaneous L885R substitution in the kinase domain Dwarsfim with short limbs and tail; decreased number in chondrocytes of the proliferative and hypertrophic regions of the growth plates

NPR-B mutations in humans Homozygous mutations associated with acromesomelic dysplasia, maroteaux type; Heterozygous mutations associated with short stature

ANP

CNP

NPR-C/ mice Increased level of available CNP Skeletal overgrowth

BNP

NPR-A

NPR-B

P

P

P

P

P

P

P

P

GTP

NPR-C

GMP

Enhanced downstream signaling

Fig. 1. Schematic representation of natriuretic peptides and their receptors with a particular focus on the CNP/NPR-B signaling system. CNP binds with high affinity to the NPR-B receptor present at the cell membrane. Thanks to its guanylyl cyclases activity, the activated receptor transduces the signal into the cell through the production of the cyclic GMP second messenger. The NPR-C molecules lack a cytosolic kinase domain and work as clearance receptors. CNP and its receptor are involved in the control of endochondral ossification. The effects of targeted or spontaneous mutations affecting this pathway at different levels both in animal models and in humans, are summarized in the rectangles

and terminal differentiation [1]. This process involves both systemic hormones and local regulators cooperating to allow harmonious and coordinated bone growth throughout the body. In this context, the structural and/or functional perturbation of some of these factors can lead to dysregulation of these pathways and to skeletal malformation syndromes. Natriuretic peptides are a family of structurally related hormones/paracrine factors involved in the modulation of cell/tissue homeostasis (regulation of blood volume and blood pressure, ventricular hypertrophy, pulmonary hypertension, fat metabolism, and long bone growth) (for review, see [2]). In mammals, three members of this family are known: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) [2]. Three single membrane-spanning

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natriuretic peptide receptors (NPRs) have been identified: NPR-A/GC-A/NPR1 and NPR-B/GC-B/NPR2 receptors show guanylyl cyclases activity, whereas NPR-C/ NPR3, lacking intrinsic enzymatic activity, is involved in controlling local concentrations of natriuretic peptides through constitutive receptor-mediated internalization and degradation (for review, see [2]) (fig. 1). The CNP is encoded by the NPPC gene located on chromosome 2q37.1. CNP signaling is mediated by interaction with the NPR2 receptor. Receptor dimerization and activation upon CNP binding trigger the intracellular signaling through generation of the cyclic GMP second messenger. The intensity and duration of the signal is modulated at different levels. One of the most important silencing mechanisms is realized through the binding of CNP to NPR3 that works as clearance receptor [3] (fig. 1). CNP and its cognate receptor are well expressed in cartilage (for review, see [4]) and several data obtained from both cultured cells and animal models have confirmed the role of CNP as regulator of cartilage homeostasis, proliferation and differentiation of osteoblasts and osteoclasts, and endochondral bone growth [4–7]. Knock-out mice for CNP or NPR-B coding genes show a marked dwarfism, with a significant reduction in the thickness of both proliferative and hypertrophic zones of the cartilage growth plates [3, 8, 9]. A similar phenotype is also observed in a spontaneous mouse model harboring the substitution of a conserved residue in the kinase domain of NPRB [8, 9]. These mice are dwarfed with short limbs and tails, due to a decreased number of both proliferative and hypertrophic chondrocytes in growth plates. Conversely, transgenic mice overexpressing the NPPC gene under the control of the Col2A1 promoter are characterized by skeletal overgrowth with expansion of both proliferative and hypertrophic zones of the cartilage growth plates [10] (fig. 1). In humans, homozygous mutations of the NPR2 gene have been found in patients affected with the acromesomelic dysplasia, Maroteaux type [11], whereas heterozygous mutations in the NPR2 gene associated with short stature have been described [12] (fig.1). So far, no mutations in the NPPC gene have been found in humans. However, recently, two independent works have described a relationship between overexpression of the NPPC gene and its product and a particular phenotype characterized by skeletal overgrowth [13, 14]. Both reports moved from the molecular characterization of different chromosomal translocations involving chromosome 2, where the NPPC gene maps, and other unrelated chromosomes. The study of balanced chromosomal rearrangements is often a powerful tool to identify genes involved in human diseases. The first work carried out by our group [13] started with the clinical observation of a girl with a marfanoid habitus, arachnodactyly of the hands and feet, very long allux, scoliosis and marked overgrowth of the long bones. The patient’s karyotype showed the presence of a de novo balanced translocation between chromosomes 2 and 7, t(2;7)(q37.1;q21.3). The molecular characterization of the translocation breakpoints has allowed focussing on NPPC on chromosome 2 and the COL1A2 gene on chromosome 7. Actually, the breakpoint on chromosome 2 caused the truncation of a

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gene, called DIS3L2, thus predicting the presence of a single functional copy of it. No functional data were available about the role of this gene. The DIS3L2 locus shows a wide and apparently nonspecific expression profile, and even though it was not possible to exclude its involvement in the genesis of the patient’s phenotype, the search for candidate genes was extended also to the adjacent genomic regions. The breakpoint was around 147 kb telomeric to the 5 end of the NPPC gene, which, given the role of the latter in the modulation of endochondral ossification, strongly attracted our attention. On the other hand, the breakpoint on chromosome 7 resulted to be located 75 kb upstream of the COL1A2 gene, which is well expressed in chondrocytes [15] and is mutated in osteogenesis imperfecta [16]. As the rearrangement was not affecting the coding sequences of these genes, we focused on a possible ‘position effect’ of the translocation influencing gene expression. The expression of COL1A2 from the translocated allele was found to be reduced without significant effect on the total collagen level. Very interestingly, the NPPC gene was overexpressed in patient’s cells compared to what observed in controls and this transcriptional upregulation correlated with a CNP plasma concentration significantly higher than in normal individuals. In the same article we described the generation of transgenic mice overexpressing NPPC in bone. These mice showed a peculiar skeletal phenotype highly reminiscent of the patient’s clinical picture. All these data allowed us to suggest that CNP overproduction was responsible for the patient’s clinical picture. Shortly after publication of our article, a second work supporting our findings was published by Moncla et al. [14]. In their article, the authors described 2 patients with a clinical phenotype very similar to that of our case and featured by postnatal overgrowth, marfanoid habitus, and arachnodactyly with abnormally long allux. Both patients were carriers of a balanced translocation involving chromosome 2 and chromosomes 8 and 13, respectively. The breakpoints on chromosome 2 were clustered around the DIS3L2 locus, as in the translocation reported in our work (fig. 2, vertical arrows). Again, a significant increased amount of NPPC transcripts was present in cells from these patients compared to control cells, supporting the hypothesis that this over-expression could be related to the observed skeletal phenotype. Also in this article, the DIS3L2 gene has not been considered a good candidate for the observed clinical phenotype because, in 1 of the 2 cases the breakpoint was outside of the coding sequence and no expression alteration was observed. On the contrary, the NPPC overexpression was common to all 3 patients [13, 14]. Concerning the molecular mechanisms leading to the enhanced NPPC expression, the hypothesis that can be drawn from these works is that the translocations separate the gene from a cis-acting long-range controlling region, with silencing activity on the NPPC transcription. Balanced chromosomal rearrangements are only rarely associated with a clinical phenotype. When it occurs, they become powerful tools in discovering potential disease genes. A chromosomal rearrangement may affect gene function in different

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A NPPC

B

C *

DIS3L2

100% 1 50% 100% 2 50% 100% 3 50% 232.543 k

232.593 k

232.643 k

232.693 k

232.743 k

232.793 k

232.843 k

232.893 k

232.943 k

Fig. 2. Comparative analysis of a 460-kb genomic region spanning the NPPC and DIS3L2 genes on chromosomes 2. The panel shows the output of the comparative genomic analysis obtained with a dedicated software available at the VISTA Genome Browser (http://genome.lbl.gov/vista/index. shtml). Conserved sequences with a percentage of identity (vertical axis) higher than 70% are represented as red peaks (noncoding sequences) and blue peaks (coding sequences) and are shown relative to their position in the human genome (horizontal axis) compared to that of the mouse (1), of the chicken (2) and of the pufferfish (3). Two significantly conserved blocks downstream the DIS3L2 gene were found (rectangle and asterisk). Vertical arrows indicate the position of the breakpoints: A and B correspond to the breakpoints found in the 2 patients described by Moncla et al. [14], and C indicates the breakpoint described in the work from our group [13].

ways, by disrupting the coding sequences thus causing a defect in gene dosage or by affecting the expression of the gene even when the latter is not located in the immediate vicinity of the breakpoint. This mechanism, also described as ‘position effect’, can be due to the separation of a gene unit from other cis-acting controlling regions that can be located even several kilobases away from the gene and that can have an important role in the modulation of its expression, with both transcriptional enhancing or silencing activity [17]. These long-range control elements can be critical for proper spatiotemporal expression of a gene or for the level of this expression and are often identifiable as highly conserved noncoding sequences through a comparative genome analysis across species [18]. However, their presence is frequently only suspected or inferred and their actual identification and localization are often due to the observation of unique cases, like the ones carrying chromosomal rearrangements that provide a good starting point for research. Regarding the attractive hypothesis that NPPC overexpression is due to separation of the gene from long-range silencing elements, this is also supported by the presence of two highly conserved noncoding sequences downstream the breakpoints of the described translocations, for which functional characterization should be performed [14] (fig. 2). In conclusion, CNP overproduction can very likely be associated with a clinical phenotype in which the predominant feature is the skeletal picture characterized by

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overgrowth and bone anomalies. CNP could also have a causative role in patients with a similar phenotype showing no gross chromosomal abnormalities. In these cases, it would be of interest to evaluate the expression of the NPPC gene (or serum concentration of CNP) and eventually look for the presence of mutations or small rearrangements affecting the functional activity of both proximal and long range control elements.

References 1

2

3

4

5

6

7

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Gilbert S: Paraxial and intermediate mesoderm; in: Developmental Biology. Osteogenesis: The Development of Bones. Sunderland, Sinauer Associates, 2003, pp 474–477. Potter LR, Abbey-Hosch S, Dickey DM: Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27:47–72. Matsukawa N, Grzesik WJ, Takahashi N, et al: The natriuretic peptide clearance receptor locally modulates the physiological effects of the natriuretic peptide system. Proc Natl Acad Sci USA 1999;96: 7403–7408. Pejchalova K, Krejci P, Wilcox WR: C-natriuretic peptide: an important regulator of cartilage. Mol Genet Metab 2007;92:210–215. Yasoda A, Ogawa Y, Suda M, et al: Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway. J Biol Chem 1998;273:11695–11700. Yasoda A, Komatsu Y, Nakao K, Ogawa Y: C-type natriuretic peptide(CNP)–a novel stimulator of bone growth formed through endochondral ossification. Nippon Rinsho 2004;62(suppl 2):77–81. Yasoda A, Komatsu Y, Nakao K, Ogawa Y: Growth promoting effect of natriuretic peptides on bones formed through endochondral ossification. Nippon Rinsho 2004;62(suppl 9):60–64. Chusho H, Tamura N, Ogawa Y, et al: Dwarfism and early death in mice lacking C-type natriuretic peptide. Proc Natl Acad Sci USA 2001;98:4016–4021. Tsuji T, Kunieda T: A loss-of-function mutation in natriuretic peptide receptor 2 (Npr2) gene is responsible for disproportionate dwarfism in cn/cn mouse. J Biol Chem 2005;280:14288–14292.

10 Yasoda A, Komatsu Y, Chusho H, et al: Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med 2004;10:80–86. 11 Bartels CF, Bukulmez H, Padayatti P, et al: Mutations in the transmembrane natriuretic peptide receptor NPR-B impair skeletal growth and cause acromesomelic dysplasia, type Maroteaux. Am J Hum Genet 2004;75:27–34. 12 Olney RC, Bukulmez H, Bartels CF, et al: Heterozygous mutations in natriuretic peptide receptor-B (NPR2) are associated with short stature. J Clin Endocrinol Metab 2006;91:1229–32. 13 Bocciardi R, Giorda R, Buttgereit J, et al: Overexpression of the C-type natriuretic peptide (CNP) is associated with overgrowth and bone anomalies in an individual with balanced t(2;7) translocation. Hum Mutat 2007;28:724–31. 14 Moncla A, Missirian C, Cacciagli P, et al: A cluster of translocation breakpoints in 2q37 is associated with overexpression of NPPC in patients with a similar overgrowth phenotype. Hum Mutat 2007;28: 1183–1188. 15 Karsenty G, Park RW: Regulation of type I collagen genes expression. Int Rev Immunol 1995;12:177– 185. 16 Marini JC, Forlino A, Cabral WA, et al: Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 2007;28:209–221. 17 Kleinjan DA, van Heyningen V: Long-range control of gene expression: emerging mechanisms and disruption in disease. Am J Hum Genet 2005;76:8–32. 18 Frazer KA, Elnitski L, Church DM, Dubchak I, Hardison RC: Cross-species sequence comparisons: a review of methods and available resources. Genome Res 2003;13:1–12.

Renata Bocciardi, PhD Laboratory of Molecular Genetics G. Gaslini Institute, Largo G. Gaslini, 5 IT–16148 Genova (Italy) Tel. +39 010 563 6797, Fax +39 010 377 9797, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 67–82

Role of Transcription Factors in Midline Central Nervous System and Pituitary Defects Daniel Kelberman ⭈ Mehul Tulsidas Dattani Developmental Endocrine Research Group, Institute of Child Health, London, UK

Abstract The anterior pituitary gland is a central regulator of growth, reproduction and homeostasis, and is the end-product of a carefully orchestrated pattern of expression of signalling molecules and transcription factors leading to the development of this complex organ secreting six hormones from five different cell types. Naturally-occurring and transgenic murine models have demonstrated a role for many of these molecules in the aetiology of combined pituitary hormone deficiency (CPHD). These include the transcription factors HESX1, PROP1, POU1F1, LHX3, LHX4, SOX2 and SOX3. The expression pattern of these transcription factors dictates the phenotype that results when the gene encoding the relevant transcription factor is mutated. The highly variable phenotype may consist of isolated hypopituitarism, or more complex disorders such as septo-optic dysplasia. Since mutations in any one transcription factor are uncommon, and since the overall incidence of mutations in known transcription factors is low in patients with CPHD, it is clear that many genes remain to be identified, and characterization of these will further elucidate the pathogenesis of these complex conditions, Copyright © 2009 S. Karger AG, Basel and also shed light on normal pituitary development

Several transcription factors involved in the embryological development of the murine pituitary appear to also be involved in human pituitary organogenesis [1–4]. Spontaneous or artificially induced mutations and gene knockouts in the mouse have led to significant insights into human pituitary disease, with the identification of human mutations in a number of genes which give rise to hypopituitary phenotypes in their respective murine orthologues. Many have been implicated in the aetiology of both murine and human hypopituitarism including Hesx1, Lhx3, Lhx4, Prophet of Pit1 (Prop1), Pou1f1 (previously called Pit-1), Sox2 and Sox3. This review will deal primarily with transcription factors implicated in the aetiology of hypopituitarism in humans as a result of the identification and characterization of mutations within these genes in patients and their respective murine orthologues.

HESX1

Given the closely linked development of the pituitary gland and forebrain during normal embryogenesis, it is not surprising that abnormalities of the two structures can be linked in human disease. One example of this is septo-optic dysplasia (SOD), often referred to as de Morsier syndrome [5], a rare, highly heterogeneous condition initially described by Reeves [6] in a 7-month-old baby with absence of the septum pellucidum and optic nerve abnormalities. The condition is defined loosely by any combination of the triad of optic nerve hypoplasia, midline neuroradiological abnormalities (such as agenesis of the corpus callosum and absence of the septum pellucidum) and pituitary hypoplasia with consequent panhypopituitarism [5–9]. Homeobox gene expressed in embryonic stem cells (Hesx1) is one of the earliest markers of the pituitary primordium, suggesting that it has a critical role in early determination and differentiation of the pituitary gland. It is also called Rpx (Rathke’s pouch homeobox) and is a member of the paired-like class of homeobox genes. The gene is first expressed during mouse embryogenesis in a small patch of cells in the anterior midline visceral endoderm as gastrulation commences. Hesx1 continues to be expressed in the developing anterior pituitary until E12, when it disappears in a spatiotemporal sequence that corresponds to progressive pituitary cell differentiation. Extinction of Hesx1 is important for activation of other downstream genes such as Prop1. Targeted disruption of Hesx1 in the mouse revealed a reduction in the prospective forebrain tissue, absence of developing optic vesicles, markedly decreased head size and severe microphthalmia reminiscent of the syndrome of SOD in humans. Other abnormalities included absence of the optic cups, the olfactory placodes and Rathke’s pouch, reduced telencephalic vesicles, hypothalamic abnormalities and aberrant morphogenesis of Rathke’s pouch. In 5% of null mutants, the phenotype was characterized by complete lack of the pituitary gland. In the majority of mutant mice, they were characterized by formation of multiple oral ectodermal invaginations and hence multiple pituitary glands. HESX1 therefore appeared to be a candidate for SOD in humans. Screening of human hypopituitary patients for mutations in HESX1 led to the identification of mutations in two siblings with SOD and subsequently other mutations have been shown to present with varying phenotypes characterized by IGHD, CPHD and SOD [10–12]. MRI revealed anterior pituitary hypoplasia, an infundibulum that is either normal or absent, and a posterior pituitary that is either eutopic or ectopic. Mutations can be dominant (n = 8) or recessive (n = 5) in humans. The former demonstrate variable penetrance. We have now screened over 800 patients with SOD and hypopituitarism, and identified mutations in less than 1% of individuals confirming the rarity of HESX1 mutations [13]. As a result of this screening we have identified a number of sequence variants, including a change of unknown functional importance in a highly conserved base in a known cis-regulatory region upstream of HESX1. Whether these variants

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contribute to the pathogenesis remains to be proven. The overall frequency of HESX1 mutations is low suggesting that mutations in other known or unknown genes contribute to this complex disorder, together with a likely contribution from environmental factors [14, 15].

SOX3

The identification of a chromosomal abnormality (46, X, inv[X][p21q27]) in a patient with mental retardation led to the identification of SOX3 as a candidate gene for X-linked mental retardation (XLMR). SOX3 lay 100kb from the Xq breakpoint, and given its expression in neuroepithelial progenitor and stem cells from an early age, was thought to be a good candidate for XLMR [16]. SOX3 (OMIM 313430) is a member of the SOX (SRY-related HMG box) family of transcription factors, which were initially identified based on homology to the conserved binding motif of the high mobility group (HMG) class, present in the mammalian sex-determining gene, SRY [17]. Approximately 20 different SOX genes have been identified in mammals and variation in homology exhibited within the HMG box between different members allows them to be grouped into different subfamilies [18]. SOX3 was among the first of the SOX genes to be cloned, and together with SOX1 and SOX2, belongs to the SOXB1 subfamily exhibiting the highest degree of similarity to SRY [17]. SOX3 is encoded by a single exon producing a transcript with a coding region of approximately 1.3 kb, mapping to chromosome Xq27. The SOX3 protein consists of a short 66 amino acid N-terminal domain of unknown function, the 79 amino acid DNA binding HMG domain and a longer C-terminal domain, containing four polyalanine stretches, shown to be involved in transcriptional activation [17, 19]. Members of the SOXB1 subfamily of genes are expressed throughout the developing central nervous system (CNS) and are some of the earliest neural markers that are believed to play a role in neuronal determination [20]. High levels of expression have also been noted in the ventral diencephalon, including the infundibulum and presumptive hypothalamus [21]. Targeted disruption of Sox3 in mice results in mutants that have a variable and complex phenotype including craniofacial abnormalities, midline CNS defects, and a reduction in size and fertility [21, 22]. Sox3 mutant mice of both sexes are born with expected frequency showing no evidence for embryonic lethality, and approximately one third of mutant mice are viable and fertile with no gross abnormalities. Heterozygous females are mosaic with respect to the mutation due to X inactivation and generally appear normal, although some display a mild craniofacial phenotype. However, approximately 43% of Sox3 null mice do not survive to weaning, and the most severely affected mice exhibit profound growth insufficiency and general weakness with craniofacial defects including overgrowth and misalignment of the front teeth and abnormality of the shape of the pinna which was completely absent in some animals [21].

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The mutants had a variable endocrine deficit, the extent of which was correlated with body weight. Pituitary levels of growth hormone (GH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) were all lower in mutant compared to wild-type mice at 2 months of age [21]. Histological analysis of the pituitary gland at this stage revealed a hypoplasic anterior lobe with the presence of an additional abnormal cleft disrupting the boundary between the anterior and intermediate lobes. Further examination of Sox3 mutant embryos revealed that Rathke’s pouch displayed an abnormally expanded and bifurcated appearance in mutant embryos which possibly results in the additional cleft observed at later stages of development and in the adult pituitary. Sox3 is not expressed in Rathke’s pouch; however, it is expressed at high levels in the ventral diencephalon including the infundibulum which provides necessary inductive signals for the formation of the anterior pituitary [4]. In Sox3 mutants, the evagination of the infundibulum was less pronounced than observed in wild-type mice and the presumptive hypothalamus thinner and shorter [21]. This suggests that the hypopituitary phenotype observed in mutant mice arises as a secondary consequence of the absence of Sox3 in the ventral diencephalon. In humans, tandem duplications involving chromosome Xq26–27 have also been identified in several pedigrees with mental retardation and hypopituitarism [23–26]. By using array comparative genomic hybridisation, Solomon et al. [26] defined a critical duplication region of 3.9 Mb between Xq26.1 and Xq27.3 containing 18 annotated transcripts including SOX3. The phenotypes of affected males with X linked hypopituitarism involving duplications within this region are variable. All affected males manifest GH deficiency and varying degrees of developmental delay or mental retardation. Some individuals have been reported to have varying combinations of deficiencies of other hormones including ACTH, TSH or gonadotrophins, and complete panhypopituitarism has been documented in some cases. Woods et al. [27] described a pedigree with two half brothers manifesting evidence of X linked hypopituitarism, in the absence of developmental delay, and harbouring a submicroscopic duplication on chromosome Xq27.1, further refining the critical interval to approximately 690 kb. The first child manifested GHD and borderline low FT4 concentrations, with hypoplasia of the lower half of the infundibulum and an abnormal corpus callosum which contained a cyst within the splenium. The second sibling manifested a more severe phenotype of combined pituitary hormone deficiency, with complete absence of the infundibulum and hypoplastic genitalia; however his corpus callosum appeared normal. Both patients had anterior pituitary hypoplasia and an undescended posterior pituitary as revealed by MRI. The duplication identified in this family is the smallest described to date encompassing SOX3 and two additional transcripts of unknown function, neither of which is expressed in the developing infundibulum [27] suggesting that the phenotype in these patients is due to the presence of an additional copy of SOX3. Further implication of SOX3 in X linked hypopituitarism comes from the identification of patients harbouring an expansion of one of the polyalanine tracts within the

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gene [16,27]. Laumonnier et al. [16] identified an in frame duplication of 33 bp occurring between nucleotides 711 to 743 and co-segregating in affected males in a large family with X linked mental retardation and GH deficiency. This mutation encodes an additional 11 alanine residues and is predicted to cause expansion of the normal polyalanine tract from 15 to 26 residues. Additionally, a second novel expansion of seven alanine residues within the same tract has been identified in three siblings of a consanguineous pedigree presenting with profound and complete panhypopituitarism in association with anterior pituitary hypoplasia, an absent or hypoplastic infundibulum and an undescended posterior pituitary. There was no evidence of mental retardation or craniofacial dysmorphism in these individuals. In vitro analysis of the SOX3 +7 alanine expansion identified by Woods et al. [27], revealed that the expansion leads to partial loss of function possibly due to impairment of nuclear localization as the mutant protein was largely excluded from the nucleus, compared to wild-type SOX3 which is predominantly localized within the nucleus of the cell [27]. Similar findings have also been shown for the mutant SOX3 protein containing the +11 alanine expansion which forms aggregates within the cytoplasm [28]. In summary, both duplications of Xq27 encompassing SOX3 and loss-of-function polyalanine expansion mutations are essentially associated with similar phenotypes, predominantly infundibular hypoplasia, suggesting that gene dosage of SOX3 is critical for normal development of the diencephalon and infundibulum and, consequently, the anterior pituitary.

SOX2

SOX2 (OMIM 184429) is also a member of the same SOXB1 subfamily as SOX3 and SOX1 (OMIM 602148). In the mouse, initial expression of Sox2 is detected at 2.5 dpc at the morula stage, and then in the inner cell mass of the blastocyst at 3.5 dpc. Later expression of Sox2, following gastrulation, is restricted to the presumptive neuroectoderm and by 9.5 dpc it is expressed throughout the brain, CNS, sensory placodes, branchial arches, gut endoderm and the esophagus and trachea [29, 30]. Homozygous loss of Sox2 results in peri-implantation lethality, whereas Sox2 heterozygous mice appear relatively normal but show a reduction in size and male fertility [31]. Further studies that have resulted in the reduction of Sox2 expression levels below 40% compared to normal levels result in anophthalmia in the affected mutants [32]. Given the observation of growth retardation and reduced fertility, we investigated the role of Sox2 in murine pituitary development, showing that a proportion of heterozygous animals manifested a variable hypopituitary phenotype, with hypoplasia and abnormal morphology of the anterior pituitary gland with concomitant reduction in levels of GH, LH, ACTH and TSH [33]. Like its murine counterpart, the human SOX2 gene is composed of a single exon encoding a 317 amino acid protein

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containing an N-terminal domain of unknown function, a DNA binding HMG domain and a C-terminal transcriptional activation domain. To date, 24 de novo intragenic heterozygous mutations have been reported [30, 33–40], in addition to 8 de novo heterozygous deletions of the entire gene and 1 case of a partial deletion [30, 34, 40, 41], as well as 3 heterozygous non-synonymous sequence changes identified in individuals who inherited the variant from a clinically unaffected parent [33, 42]. The phenotypes include bilateral anophthalmia or severe microphthalmia with additional abnormalities including developmental delay, learning difficulties, oesophageal atresia and genital abnormalities [30, 34–37]. SOX2 mutations were also associated with anterior pituitary hypoplasia on MRI and hypogonadotrophic hypogonadism, which resulted in the absence of puberty in 9/10 patients studied by our group, and genital abnormalities in males [33, 43]. All affected individuals exhibited learning difficulties with other variable manifestations including hippocampal abnormalities and defects of the corpus callosum on MRI, esophageal atresia, hypothalamic hamartoma and sensorineural hearing loss [33]. The mutations were associated with significant loss of function that included loss of DNA binding, nuclear localization, and transcriptional activation, suggesting that these phenotypes arise as a result of haploinsufficency of SOX2 in development. More recently, Sato et al. [38] have reported an additional patient with a missense mutation in the HMG domain (L75Q) resulting in decreased DNA binding affinity of the mutant protein. The affected individual manifested unilateral right sided anophthalmia and isolated hypogonadotrophic hypogonadism, with a normal anterior pituitary and normal mental development, further supporting a critical role for SOX2 in the regulation of correct gonadotrophin production in addition to eye development. We have also shown that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to efficiently repress this activity [43]. Given the critical role of Wnt signalling in the development of most of these tissues, our data suggest that a failure to repress the WNT-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-offunction mutations in SOX2. Furthermore, we have shown that SOX2 is expressed throughout the human brain including the developing hypothalamus as well as Rathke’s pouch, the developing anterior pituitary, the trachea and oesophagus, the inner ear, and the eye. SOX2 expression patterns in human embryonic development therefore support a direct involvement of the protein during development of tissues affected in individuals harbouring heterozygous SOX2 mutations [43].

LHX3/LHX4

Lhx3 is a member of the LIM family of homeobox genes which are characterized by the presence of a unique cysteine/histidine-rich zinc-binding LIM domain. The protein contains two such tandemly repeated LIM motifs between the N-terminus and

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the homeodomain, which are likely to be involved in protein-protein interactions [44, 45]. Lhx3 is one of the earliest transcription factors expressed within the developing pituitary, initially detectable with strong uniform expression in Rathke’s pouch. Expression is maintained in the pouch and is subsequently restricted to fields fated to form the anterior and intermediate lobes. By 16.5 dpc, the gene is expressed in all regions of the developing anterior and intermediate pituitary, but not the posterior gland, and continued expression is essential for the establishment of hormone producing cell types. Expression persists throughout development and is also detected in the adult pituitary suggesting a role in maintenance of one or more of the mature anterior pituitary cell types [44, 46]. In addition to the pituitary, Lhx3 is also detected transiently in regions of the developing spinal cord, prior to neural tube closure, and later within restricted regions of the hindbrain, in addition to cells in immediate proximity to the otic vesicles. Mice with a targeted homozygous disruption of Lhx3 die shortly after birth, although the cause of death is unknown, and exhibit pituitary aplasia, suggesting an essential role of Lhx3 in differentiation and proliferation of anterior pituitary cell lineages. Although Rathke’s pouch is initially formed in Lhx3 null mice, a failure of proliferation and growth results in a lack of the anterior and intermediate lobes of the pituitary with depletion of all hormone-producing cell types except corticotrophs, which differentiate and express pro-opiomelanocortin (POMC), but fail to proliferate [47, 48]. Homozygous mutations in LHX3 (OMIM 600577) have currently been identified in twelve patients from seven unrelated consanguineous families, all of which result in loss of LHX3 function [49–53]. The patients presented with an endocrine phenotype similar to that observed in individuals with PROP1 mutations with a deficit in all anterior pituitary hormones except ACTH. This was additionally associated in 9/12 patients with a short rigid cervical spine with limited head rotation and trunk movement. As with PROP1-deficient patients, pituitary morphology is variable between patients with LHX3 mutations, with two patients from one family exhibiting small anterior pituitaries with a normal posterior pituitary and midline structures on MRI. However, an additional individual from an unrelated family demonstrated a markedly enlarged anterior pituitary that was not evident in a previous MR scan performed ten years previously [49]. Additionally, Bhangoo et al. [51] recently reported a further patient with a hypointense lesion in the anterior pituitary consistent with a microadenoma. Lhx4 is closely related to Lhx3 and is expressed in specific fields of the developing brain and spinal cord. Similar to Lhx3, Lhx4 is initially expressed throughout the invaginating Rathke’s pouch, however subsequent expression is transient and restricted to the future anterior lobe, whereas Lhx3 expression is maintained throughout the whole pouch. A similar pattern of transient expression of LHX4 (OMIM 602146) in the developing pituitary and spinal cord, with continuous expression of LHX3, has also been observed in human development [52]. Null mutations of Lhx4 do not prevent pouch formation; however the pouch is defective with reduced numbers of the

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various anterior pituitary cell types. Lhx3–/–, Lhx4–/– double mutant mice show a more severe phenotype than either single mutant with an early arrest of pituitary development; additionally a single normal copy of either Lhx3 or Lhx4 in murine development is sufficient for formation of a definitive pouch, thereby suggesting that these two genes may act in a redundant manner during early pituitary development [54]. Mutational analysis of the first reported patient, with CPHD (GH, TSH and ACTH deficiency), revealed a heterozygous intronic mutation in LHX4 [55]. MR imaging revealed anterior pituitary hypoplasia, an undescended posterior pituitary, an absent pituitary stalk, a poorly formed sella and pointed cerebellar tonsils. The second patient presented with a similar phenotype but also had additional prolactin, LH and FSH deficiencies, Chiari malformation and respiratory distress syndrome and was found to have a heterozygous missense mutation (P366T) in exon 6, which was present in the LIM4 specific domain. [56]. More recently, Pfaeffle et al. [53] have reported three novel heterozygous mutations (A120P, L190R, R84C) in 3 unrelated families. All patients had evidence of variable hypopituitarism between and within families and a hypoplastic anterior pituitary on neuroimaging. However, an undescended posterior pituitary was not observed in patients of the first family, two of whom demonstrated pituitary cysts. Mutations in LHX4 are rare and the resultant phenotype in patients with mutations suggests that LHX4 tightly coordinates brain development and skull shape in qddition to pituitary development.

PROP1

Prop1 is a paired-like homeodomain transcription factor, expressed exclusively within the embryonic pituitary. Expression is first detected in the dorsal portion of the murine Rathke’s pouch in a region overlapping the expression domain of Hesx1. Maximal expression of Prop1 is achieved at 12 dpc in the full caudomedial area of the developing anterior pituitary followed by a marked decrease, becoming undetectable by 15.5 dpc [57]. The Ames dwarf (df) mouse harbours a naturally occurring serine to proline (S83P) substitution within the homeodomain of Prop1, resulting in a mutant protein with an 8-fold lower DNA binding affinity than wild-type Prop1 [57]. Homozygous Ames dwarf mice exhibit severe proportional dwarfism, hypothyroidism and infertility and although early pituitary development is similar to wild-type mice, the emerging anterior pituitary gland is reduced by about 50%, displaying an abnormal looping appearance [58]. The adult Ames dwarf mouse exhibits GH, TSH and prolactin deficiency resulting from a severe reduction of somatotroph, lactotroph and caudomedial thyrotroph lineages with approximately 1% of the normal complement of each cell type. Additionally, these mice have reduced gonadotrophin expression correlating with low LH and FSH plasma levels [57–59]. Following the identification of Prop1 as the gene underlying the Ames dwarf phenotype in mice, Wu et al. [60] reported the first mutations in PROP1 (OMIM 601538)

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in human patients with GH, TSH and PRL deficiency in addition to reduced gonadotrophins and a failure to enter puberty spontaneously. To date, 22 distinct mutations have been identified in more than 170 patients, suggesting that PROP1 mutations are the most common cause of CPHD reported, accounting for approximately 50% of familial cases [61, 62], although the incidence in sporadic cases is much lower [63]. All affected individuals exhibit recessive inheritance, and, with one exception, all mutations identified to date involve the DNA binding homeodomain, which is highly conserved between mouse and human sharing 91% identity at the nucleotide level [64]. The majority of these mutations are predicted to result in complete loss of function of the protein by ablating DNA binding and transcriptional activation. However, in vitro analysis has shown that some missense mutations retain partial activity [60, 65, 66]. By far the most common mutation (50–72% of all familial PROP1 mutations) [61, 62, 67], detected in multiple unrelated families from several different countries, is a 2-bp deletion among three tandem GA repeats (296-GAGAGAG-302) within exon 2 resulting in a frameshift at codon 101 and the introduction of a termination codon at position 109 (often referred to as S109X), and probably represents a mutational hot spot within the gene, rather than a single common founder mutation [62]. Recently, Reynaud et al. [68] reported the first PROP1 mutation downstream of the homeodomain involving a substitution of a tryptophan residue for a stop codon at position 194 (W194X) in the transactivation domain, with the resultant mutant protein showing only 34% activity compared with wild type PROP1. Recessive inheritance of mutations in PROP1 is typically associated with GH, TSH, PRL and gonadotrophin deficiencies, although the time of initiation and severity of pituitary hormone deficiencies is highly variable. Most patients present with early onset GH deficiency and growth retardation; however normal growth in early childhood has been reported in a patient who attained a normal final height without GH replacement therapy [69]. TSH deficiency is also highly variable and has been reported as the first presenting symptom in some cases, while others show delayed TSH deficiency which may not be present at birth [61, 70–72]. Individuals with PROP1 mutations exhibit normal ACTH/cortisol levels in early life but often demonstrate an evolving cortisol deficiency that is strongly and significantly correlated with increasing age [72–76]. However, patients as young as 6–7 years have also been described with cortisol deficiency [76, 77]. Although Prop1 is essential for the differentiation of gonadotrophs in fetal life, the spectrum of gonadotrophin deficiency is again extremely variable in patients with PROP1 mutations. Clinical variability can range from hypogonadism with complete lack of pubertal development to reports of spontaneous, albeit often delayed, onset of puberty with subsequent development of gonadotrophin deficiency requiring hormone replacement [61, 70, 71, 74]. The pituitary morphology in patients with PROP1 mutations is also highly variable; most individual reports have documented a normal pituitary stalk and posterior lobe, with a small or normal size anterior pituitary gland on MRI. However, in some

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cases, an enlarged anterior pituitary gland has been reported [60, 72, 78]. Longitudinal analyses of anterior pituitary size over time have revealed that a significant number of patients demonstrated pituitary enlargement in early childhood with subsequent regression and involution; thus ensuing MRI in older patients usually demonstrates anterior pituitary hypoplasia [75, 79]. The pituitary enlargement consists of a mass lesion interposed between the anterior and posterior lobes, possibly originating from the intermediate lobe [79]. Turton et al. [63] recently demonstrated that the mass can wax and wane in size prior to eventual involution. To date, the underlying mechanism for the mass remains unknown. There has been only one report of a biopsy of the ‘tumour’, and the histology was non-specific with the presence of amorphous material with no signs of apoptosis, and no recognisable cell types [80]. Consequent to the highly variable phenotype associated with PROP1 mutations, no genotypephenotype correlation has been identified; furthermore phenotypic differences have been reported in siblings with identical mutations [70]. The evolving nature of hormone insufficiencies in patients with PROP1 mutations suggests a progressive decline in the anterior pituitary axis, indicating a need for continual monitoring of patients for the development of hormone insufficiencies that may not be apparent at initial presentation.

POU1F1

POU1F1 (OMIM 173110; previously known as PIT1) is a pituitary-specific transcription factor belonging to the POU homeodomain family of transcription factors (named after the genes PIT1, OCT1 and unc-86) characterized by a highly conserved DNA binding domain consisting of a POU-specific domain and a POU homeodomain. In the mouse, Pou1f1 is expressed relatively late during pituitary development (14.5 dpc), and expression persists throughout post-natal life and adulthood, restricted to the anterior pituitary lobe [81]. Pou1f1 is essential for the development of somatotroph, lactotroph and thyrotroph cell lineages in the anterior pituitary [82], and for the subsequent expression of the GH-1 (OMIM 139250), prolactin (PRL; OMIM 176760) and TSH-β (OMIM 188540) genes between 15.5 and 17 dpc [83]. Two naturally occurring murine models have shed light on the role of Pou1f1 in normal pituitary development. In the Snell dwarf (dw) mouse, a recessive point mutation (W261C) results in the absence of somatotrophs, lactotrophs and thyrotrophs [84]. A similar phenotype results in the Jackson dwarf mouse (dwJ) that harbours a recessive null mutation due to rearrangement of Pou1f1. Pou1f1-binding sites have also been found in the GHRHR (OMIM 139191) and the Pou1f1 gene itself [82, 85], and autoregulation is required to sustain gene expression once the Pou1f1 protein has reached a critical threshold [86]. The first mutation within POUIFI was identified by Tatsumi et al. [87] in a child with GH, prolactin and profound TSH deficiency caused by homozygosity for a

76

Kelberman · Dattani

Table 1. Human mutations causing abnormal hypothalamo-pituitary development and function Gene

Phenotype

Inheritance

Combined pituitary hormone deficiency POU1F1 GH, TSH, prolactin deficiencies; usually severe; small or recessive, normal AP dominant PROP1 GH, TSH, LH, FSH, prolactin deficiencies; evolving recessive ACTH deficiency; small, normal or enlarged AP Specific syndrome HESX1 LHX3

LHX4

SOX3 SOX2

IGHD, CPHD, SOD; AP(H), EPP, absent infundibulum, ACC CPHD (GH, TSH, LH, FSH, prolactin deficiencies), short neck, limited rotation; small, normal or enlarged AP, microadenoma of pituitary, short cervical spine CPHD (GH, TSH, ACTH, LH, FSH deficiencies); small AP, normal PP or EPP, pituitary cysts, cerebellar abnormalities IGHD and mental retardation, panhypopituitarism; AP(H), infundibular hypoplasia, EPP hypogonadotrophic hypogonadism; AP(H), bilateral anophthalmia/microphthalmia, abnormal corpus callosum, learning difficulties, oesophageal atresia, sensorineural hearing loss

recessive, dominant recessive

dominant

X-linked de novo

AP(H) = Anterior pituitary (hypoplasia); EPP = ectopic posterior pituitary; ACC = agenesis of corpus callosum.

nonsense mutation within the gene. The majority of mutations identified in POU1F1 to date are recessive; however, in addition a number of heterozygous point mutations have been reported [88]. Of these, the amino acid substitution R271W appears to be a ‘hotspot’ for POU1F1 mutations [88], and has been identified in several unrelated patients of different ethnic backgrounds. When co-transfected with wild-type POU1F1, this mutant protein prevented transcriptional activation by the wild-type protein acting as a dominant negative [89], although this has been recently disputed [90]. The spectrum of hormone deficiency can vary in patients with POU1F1 mutations; GH and prolactin deficiencies generally present early in life, however TSH deficiency can be highly variable with presentation later in childhood [91, 92]. We have recently described a POU1F1 mutation in a 21-year-old with GH and prolactin deficiency who has normal thyroid function to date [93]. Magnetic resonance imaging demonstrates a small or normal anterior pituitary with a normal posterior pituitary and infundibulum, and no midline abnormalities. Since the first report, a total of 27 POU1F1 mutations have been described including 22 recessive and 5 dominant mutations in over

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77

60 patients originating from 19 different countries, all of which have been associated with a broadly similar phenotype of GH, TSH and prolactin deficiency [94].

Conclusions

Several transcription factors and signalling molecules are critical for cell differentiation and proliferation in the pituitary gland at a very early stage of gestation. The mouse has served as an excellent model for understanding the genetic basis of congenital hypopituitarism in humans, although the correlation between mouse and human disease phenotypes is variable. This candidate gene approach, based on mouse studies, had led to the identification of several human mutations that disrupt hypothalamopituitary development resulting in specific patterns of hormone dysfunction. Establishing the genotype can aid in the management of individual patients with hypopituitarism. For example, a patient with an identified PROP1 mutation exhibiting an enlarged anterior pituitary may be at risk of visual impairment due to anterior pituitary hyperplasia; however a number of reports in individuals with PROP1 mutations have shown the enlarged anterior pituitary to undergo spontaneous involution. Careful monitoring of the anterior pituitary in such cases may prevent the patient undergoing further invasive procedures. Additionally, identification of a mutation within POU1F1 predicts that cortisol and gonadotrophin secretion will remain normal in the patient. Identification of the genotype can also aid in genetic counselling and early diagnosis, particularly in autosomal dominant POU1F1 mutations. However, no genetic aetiology has been established to date in most patients with hypopituitarism. Given that a number of these patients may represent familial cases, it is clear that many genes implicated in hypopituitarism remain to be identified. Mapping and identification of the underlying causative mutations in these rare familial cases will help to identify novel genes for mutational screening in the more common sporadic forms of the condition.

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86 Rhodes SJ, Chen R, DiMattia GE, Scully KM, Kalla KA, Lin SC, Yu VC, Rosenfeld MG: A tissue-specific enhancer confers Pit-1-dependent morphogen inducibility and autoregulation on the pit-1 gene. Genes Dev 1993;7:913–932. 87 Tatsumi K, Miyai K, Notomi T, Kaibe K, Amino N, Mizuno Y, Kohno H: Cretinism with combined hormone deficiency caused by a mutation in the PIT1 gene. Nat Genet 1992;1:56–58. 88 Cohen LE, Wondisford FE, Salvatoni A, Maghnie M, Brucker-Davis F, Weintraub BD, Radovick S: A ‘hot spot’ in the Pit-1 gene responsible for combined pituitary hormone deficiency: clinical and molecular correlates. J Clin Endocrinol Metab 1995;80:679– 684. 89 Radovick S, Nations M, Du Y, Berg LA, Weintraub BD, Wondisford FE: A mutation in the POUhomeodomain of Pit-1 responsible for combined pituitary hormone deficiency. Science 1992;257: 1115–1118. 90 Kishimoto M, Okimura Y, Fumoto M, Iguchi G, Iida K, Kaji H, Chihara K: The R271W mutant form of Pit-1 does not act as a dominant inhibitor of Pit-1 action to activate the promoters of GH and prolactin genes. Eur J Endocrinol 2003;148:619–625. 91 Pfaffle RW, DiMattia GE, Parks JS, Brown MR, Wit JM, Jansen M, Van-der-Nat H, Van-den-Brande JL, Rosenfeld MG, Ingraham HA: Mutation of the POU-specific domain of Pit-1 and hypopituitarism without pituitary hypoplasia. Science 1992;257:1118– 1121. 92 Pfaffle RW, Martinez R, Kim C, Frisch H, Lebl J, Otten B, Heimann,G: GH and TSH deficiency. Exp Clin Endocrinol Diabetes 1997;105(suppl 4):1–5. 93 Turton JPG, Reynaud R, Mehta A et al: Novel mutations within the POU1F1 gene associated with variable combined pituitary hormone deficiency. J Clin Endocrinol Metab 2005;90:4762–4770. 94 Cohen LE, Radovick S: Molecular basis of combined pituitary hormone deficiencies. Endocr Rev 2002; 23:431–442.

Mehul Tulsidas Dattani, MD Developmental Endocrine Research Group Institute of Child Health, 30 Guilford Street London WC1N 1EH (UK) Tel. +44 0207 905 2657, Fax +44 0207 404 6191, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 83–94

Developmental Abnormalities of the Posterior Pituitary Gland Natascia di Iorgi ⭈ Andrea Secco ⭈ Flavia Napoli ⭈ Erika Calandra ⭈ Andrea Rossi ⭈ Mohamad Maghnie Departments of Pediatrics and Neuroradiology (A.R.), IRCCS G, Gaslini Institute, University of Genova, Genova, Italy

Abstract While the molecular mechanisms of anterior pituitary development are now better understood than in the past, both in animals and in humans, little is known about the mechanisms regulating posterior pituitary development. The posterior pituitary gland is formed by the evagination of neural tissue from the floor of the third ventricle. It consists of the distal axons of the hypothalamic magnocellular neurones that shape the neurohypophysis. After its downward migration, it is encapsulated together with the ascending ectodermal cells of Rathke’s pouch which form the anterior pituitary. By the end of the first trimester, this development is completed and vasopressin and oxytocin can be detected in neurohypophyseal tissue. Abnormal posterior pituitary migration such as the ectopic posterior pituitary lobe appearing at the level of median eminence or along the pituitary stalk have been reported in idiopathic GH deficiency or in subjects with HESX1, LHX4 and SOX3 gene mutations. Another intriguing feature of abnormal posterior pituitary development involves genetic forms of posterior pituitary neurodegeneration that have been reported in autosomal-dominant central diabetes insipidus and Wolfram disease. Defining the phenotype of the posterior pituitary gland can have significant clinical implications for management and counseling, as well as providing considerable insight into normal and abnormal mechanisms of posterior pituitary development in Copyright © 2009 S. Karger AG, Basel humans.

The molecular mechanisms and roles of transcription factors in anterior pituitary development are now much better understood than in the past, both in animals and in humans, but little is known about the mechanisms that regulate posterior pituitary development. The anterior, intermediate and posterior lobes of the pituitary gland develop from separate embryonic cell lineages: the oral ectoderm and neural ectoderm, respectively. Pituitary organogenesis begins when an area of oral ectoderm at the roof of the presumptive oral cavity invaginates upwards to form the Rathke’s pouch, which

Timeline Prenatal time scale (months)

Lateral ventricles

SIM1 ARNT2

OTP

Blastocyst

Ventricular layer

BRN2 1 SS

PVN

Embryo

Third Movement path ventricle of cellular migration

TRH

CRH

AVP

OT

Differentiation of neurones involves cascades of transcription factors which progressively define their phenotype

2

PVN

Lateral ventricles

SON Caudate nuclei Thalamus

3 Fetus

4

SON Third Hypothalamus ventricle Indicates direction of migration of cells from the ventricular layer, through the PVN to their resting place in the SON

9 Birth

Optic chiasm

Anterior pituitary

Mamillary body Posterior pituitary

Completion of the formation of the posterior pituitary involves the development of axonal projections from the PVN and SON through the neural stalk, terminating in the posterior pituitary lobe

Fig. 1. Timeline of the development of the posterior pituitary neurone. SIM1, ARNT2, OTP and BRN2 are genes involved in the cascade of transcription factors. PVN = Paraventricular nuclei; SON = supraoptic nuclei: SS = synthesizing somatostatin; TRH = thyrotropin-releasing hormone; CRH = corticotrophin-releasing hormone; AVP = arginine vasopressin; OT = oxytocin. With permission.

eventually becomes the anterior and intermediate lobes of the gland. The posterior pituitary evolves from an infundibulum that develops by downward evagination of the dorsal presumptive diencephalon soon after Rathke’s pouch begins to extend upward. The two structures maintain close contact while cells migrate from the mesoderm and neural crest into the space between the presumptive brain and oral cavities. The ventral wall of Rathke’s pouch becomes the anterior lobe whereas the posterior wall of the pouch develops into the pars intermedia [1]. The following sections of this article will focus on the most important aspects of diseases involving abnormal posterior pituitary gland development.

Abnormal Development of the Posterior Pituitary Lobe

The hypothalamo-neurohypophysis consists of magnocellular neurons that produce the peptide hormones vasopressin and oxytocin. The cell bodies are located in the

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paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus, and axons project to the neurohypophysis where the hormones are secreted into the bloodstream. During embryogenesis, neuroepithelial cells from the lining of the third ventricle migrate to the walls of the third ventricle where they mature into PVN. Some cells continue to migrate laterally towards and above the optic chiasm to form the SON. Their unmyelinated axons traverse the basal hypothalamus, form the neural stalk and terminate at the floor of the third ventricle and in the median eminence. The early differentiation of these cell lineages has recently been made clearer through an understanding of the role of transcription factors in hypothalamic development [2]. The SIm1, ARNt2, OTP, and BRN2 genes appear to be involved in the cascade of transcription factors implicated in the development of the neuroendocrine hypothalamus leading to the completion of posterior pituitary development by the end of the first trimester (fig. 1), when vasopressin and oxytocin can be detected in neurohypophyseal tissue [3]. Null mutation of UNcx4.1 showed a phenotype consisting of an ectopic localization that extends vasopressinergic axons from PVN and SON; these axons do not halt at the proper position in the neurohypophysis, but instead grow into the anterior pituitary lobe [4]. In humans, a similar picture has been reported in association with cerebral malformations [5]. In a recent study, a Hes1-null pituitary gland was revealed to be reduced in size but was otherwise morphologically normal compared with the control. Indeed, in Hes1-Hes5 double-mutant mice, the evagination of the infundibulum was affected and the neurohypophysis was lost compared to both the wild-type and Hes1-null mice, suggesting that both Hes genes are essential for the formation of the neurohypophysis [6]. In the nervous system, both Hes1 and Hes5 are essential for the regulation of neural stem cells, while in the endocrine system, Hes1 controls pancreatic cell differentiation: indeed, an analysis of mice deficient for Prop1 has indirectly implicated involvement of the Notch pathway in pituitary development [7].

Relationship between Posterior Pituitary Development and Pituitary Diseases

Abnormal posterior pituitary development can be subdivided into two main categories (fig. 3). The first is associated with a migration defect and the second with neurodegeneration of the hypothalamic PVN and SON nuclei. Developmental abnormality of the posterior pituitary will lead to an ectopic posterior pituitary (EPP) at the median eminence or along the pituitary stalk with partial or complete pituitary stalk agenesis with or without additional central nervous system malformations where the genetic and the idiopathic forms are closely linked and might have a common origin [8]. On the other hand, the neurodegeneration of the hypothalamic nuclei will lead to posterior pituitary dysfunction and central diabetes

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85

Posterior pituitary pathologies

Developmental abnormality–migration ectopic posterior pituitary

Neurodegenerative neurones

Wolfram syndrome Autosomal-dominant FNDI

Partial or complete agenesis pituitary stalk CNS malformations

Partial or complete agenesis pituitary stalk

Astrocytoma Choristoma (granular cell tumor)

Genetic syndromes idiopathic

Neurohypophyseal germinoma

Posterior pituitary function

Fig. 2. Tree diagram showing the two main categories of developmental anomalies of the posterior pituitary gland.

insipidus. Whether posterior pituitary function is affected in patients with developmental abnormalities of the posterior pituitary remains uncertain.

Abnormal Posterior Pituitary Development and Phenotype Craniofacial dysmorphisms and eye abnormalities are often manifestations of major developmental syndromes and can give useful clues about possible defects of the brain and/or hypothalamus and pituitary gland [9–10]. Some facial malformation syndromes including septo-optic dysplasia (SOD) and Pallister-Hall are easy to recognize by visual and/or physical examination. Indeed, the diagnosis of hypopituitarism in childhood can sometimes be straightforward when short stature and persistent growth failure are associated with frontal bossing, mid-facial hypoplasia and truncal adiposity; however, this presentation tends to be the exception rather than the rule and thus the clinical phenotype may not be particularly impressive. Recent study shows, however, that patients with an EPP at magnetic resonance imaging (MRI) had significantly higher canthal index CI values (the relative distance between the eyes; hypertelorism is generally defined as CI >42, and hypotelorism as CI <38) compared

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a

b

d

e

c

f

Fig. 3. a Normal pituitary gland appearance on sagittal T1-weighted images. The posterior pituitary hyperintensity (PP, arrowhead), anterior pituitary lobe (AP, thick arrow), pituitary stalk (PS, thin arrow) and median eminence (ME) are clearly visible. b Case with MPHD, small pituitary sella, anterior pituitary hypoplasia (thick arrow), absent pituitary stalk and ectopic posterior lobe at the of the median eminence (arrowhead). c Case with MPHD, small pituitary sella, anterior pituitary hypoplasia (thick arrow), and ectopic posterior lobe at the distal end (arrowhead) of the hypoplastic pituitary stalk (thin arrow). d Case with isolated GHD, small pituitary sella, anterior pituitary hypoplasia and ectopic posterior lobe within the middle third (arrowhead) of pituitary stalk. e Case with isolated GHD, small pituitary sella, anterior pituitary hypoplasia and ectopic posterior lobe at the distal end (arrowhead) of pituitary stalk. f Case 7 with evolving pituitary hormone deficiency, small pituitary sella, anterior pituitary hypoplasia and ectopic posterior lobe extended (arrowhead) within the pituitary stalk.

to patients who had in situ posterior pituitary [11]. Ectopic posterior pituitary has occasionally been reported in several other syndromes as well [12–17] (table 1).

Genes and Ectopic Posterior Pituitary Ectopic posterior pituitary gland associated with anterior pituitary dysfunction has been reported in a variety of naturally occurring mutations of transcription factors involved in pituitary development. In particular, human mutations of HESX1, LHX4 and SOX3 can give rise to abnormal anterior pituitary organogenesis and posterior pituitary migration with or without additional central nervous system and/or extrapituitary abnormalities (table 2).

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Table 1. Syndromes associated with developmental abnormalities of the posterior pituitary gland Septo-optic-dysplasia [1] Dubowitz syndrome [15] Poland’s syndrome [12] Worster-Drought syndrome [16] Arthrogryposis multiplex congenital [13] Situs inversus totalis [17] Fanconi’s anemia [14]

Table 2. Gene mutations associated with abnormal development of the posterior pituitary Gene inheritance

Hormone deficiencies

HESX1 AR/AD/v.p.

GH

LHX4 AD

GH

SOX3 X-linked R

GH

± PRL

PRL

Pituitary phenotypes

Associated abnormalities

±TSH

± LH/ FSH

± ACTH normal/aplasia/ SOD AP hypoplasia SOD variants EPP/AP hypoplasia

TSH

LH/FSH ACTH (?)

pituitary cysts/ AP hypoplasia/ EPP*

Arnold-Chiari I

TSH

LH/FSH ACTH

AP hypoplasia/ EPP

mental retardation (variable)

AR = Autosomal recessive; AD =autosomal dominant; v.p. = variable penetrance; AP = anterior pituitary; EPP =ectopic posterior pituitary; SOD =septo-optic dysplasia; ONH= optic nerve hypoplasia; CNS = central nervous system. *Posterior pituitary in situ has been recently reported.

HESX1 Various pituitary phenotypes have been reported in association with a HESX1 mutation and, interestingly, HESX1 was the first gene mutation described with EPP (a common finding in idiopathic GHD). However, the majority of a cohort of more than 500 patients with a variety of pituitary gland disorders (of whom approximately 200 were candidates for SOD variants) showed no mutation when screened [1, 18]. The pituitary phenotype in HESX1 mutations has been reported to be associated with agenesis of the corpus callosum, hypoplasia of the optic nerves, pituitary hypoplasia and EPP. In other cases, MRI examination has evidenced unilateral hypoplasia of the optic nerve associated with a normal or hypoplastic pituitary gland in patients with isolated GH deficiency or with panhypopituitarism. Aplasia of the anterior pituitary and optic nerve coloboma, as well as anterior pituitary aplasia in the absence of optic nerve abnormalities, have also been reported recently [8, 18].

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LHX4 Three reports have described patients with LHX4 gene mutations. In the first, analysis of a consanguineous family with members exhibiting multiple pituitary hormone deficiency (MPHD), short stature, small sella turcica, hypoplastic anterior pituitary, and cerebellar defects revealed a heterozygous mutation in an intron of LHX4 [19]. These patients presented with deficiencies in GH, TSH, and ACTH (LH and FSH were not investigated). A second heterozygous mutation (P366T) affecting a residue in the carboxyl terminus of LHX4 was associated with deficiencies of GH, prolactin, TSH, LH, FSH, ACTH, a hypoplastic anterior lobe, an ectopic posterior pituitary, a poorly developed sella turcica, Arnold-Chiari malformation, and respiratory distress syndrome [20]. In a recent study of 5 patients, three types of heterozygous missense mutations in LHX4 were identified. The patients have GH deficiency and some also display reductions in TSH, LH, FSH, or ACTH. Three patients displayed in situ posterior pituitary and two had ectopic posterior pituitary while the anterior pituitary was hypoplastic in 3 and normal in one; pituitary cysts were found in 2 patients [21]. SOX3 Recent studies implicate SOX3, a single exon gene closely related to SRY, in the etiology of X-linked hypopituitarism in both humans and in mice. The phenotype of Sox3 mutant mice is variable and complex, with abnormalities throughout the hypothalamic-pituitary-gonadal axis. Indeed, in humans, mutations of SOX3 and duplications at Xq26–27 are implicated in a syndrome of X-linked hypopituitarism and mental retardation. Both Sox3 overdosage and underdosage cause clinical and endocrine hypopituitarism-related conditions associated with EPP, and abnormality of the infundibulum which suggests that gene dosage of SOX3 is critical for normal development of the diencephalon and infundibulum, and consequently of the anterior pituitary [1].

Idiopathic Forms of Ectopic Posterior Pituitary Patients with idiopathic GH deficiency can present with a variety of pituitary MRI features including: normal pituitary gland size, anterior pituitary hypoplasia or empty sella (these 3 conditions present with normal location of the posterior pituitary gland and normal pituitary stalk connecting the hypothalamus to the pituitary gland); and/ or moderate (pituitary height of 2–3 mm) to severe (pituitary height <2 mm) pituitary hypoplasia and sella turcica associated with EPP at the level of the median eminence or along the stalk projection up to its distal extremity with partial or complete pituitary stalk agenesis. In particular, the classic triad of EPP, pituitary stalk agenesis and anterior pituitary stalk is the most common variant of MRI abnormality in patients with GH deficiency [22]. These features have been reported with or without additional malformations of the central nervous system [23], including malformations

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a b • • • • • •

Arnold-Chiari type I Tentorial anomaly Septum pellucidum agenesis Septo-optic dysplasia Cortical dysplasia Absence of internal carotid artery

• Arnold-Chiari type II • Vermis dysplasia • Periventricular heterotopia • Corpus callosum dysgenesis • Basilar impression • Arachnoid cyst • Syringomyelia

c

Fig. 4. Small arrows showing ectopic posterior pituitary (EPP) in figures (a) and (b). Long arrow shows Arnold-Chiari I malformation in figure b. Arrows indicate syringomyelia in figure c.

like Arnold-Chiari I, Arnold-Chiari II, agenesis of the septum pellucidum, SOD/SOD variants, vermis dysplasia, syringomyelia, absence of internal carotid artery, dysgenesis of the corpus callosum, arachnoid cysts, and tentorium anomalies with basilar impression (fig. 4). The pathogenetic mechanisms involved in the development of EPP in idiopathic GH deficiency are not yet completely understood. However, a high percentage of birth trauma or breech delivery has been reported in patients with GH deficiency and ectopic posterior pituitary. It is now generally agreed that breech presentation and premature delivery are probably the result, rather than the cause of most congenital hypothalamic-pituitary defects. Two thirds of these patients are born with no reported adverse perinatal events, with cephalic delivery in approximately 50% and caesarean section in 15% of cases. The precise cause of the high frequency of breech births in cases with EPP remains unclear, though a hypothesis of fetal hypotonia secondary to anatomical and pituitary dysfunction leading to breech presentation appears to be the most plausible explanation [23–25].

Endocrine Consequences Related to Abnormal Posterior Pituitary Development

Published data show a varying correlation between anomalies of posterior pituitary development as observed at MRI scan and patient endocrine function. In particular, MPHD has been shown to be associated with ectopic posterior pituitary and anterior pituitary hypoplasia at a high rate, ranging between 74 and 100% of cases. Isolated GH deficiency, on the other hand, is associated with these particular MRI features at a lesser rate, ranging between 2 and 68% [8, 22, 23]. A detailed study of the pituitary

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a

b

(a-b) Small EPP (thick arrow) Severe AP hypolasia (thin arrow) Patient with MPHD

c

d

e

f

(c-d) Large EPP (thick arrow) Mild AP hypolasia (thin arrow) Patient with GH deficiency

(e-f ) XXL EPP (thick arrow) Bordeline AP size (thin arrow) Patient with GH deficiency

Fig. 5. Different sizes of ectopic posterior pituitary (EPP) and anterior pituitary (AP) are represented in both sagittal (a, c, e) and coronal (b, d, f) T1-weighted MRI scans. a, b patient with MPHD. Thick arrow = small EPP; thin arrow = severe AP hypoplasia. c, d Patient with GH deficiency. Thick arrow = Large EPP; thin arrow = mild AP hypoplasia. e, f Patient with GH deficiency. Thick arrow = XXL EPP; thin arrow = borderline AP size.

stalk should be carried out after the administration of contrast medium injection using gadopentetate dimeglumine (Gd-DTPA). A finding of vascular component of the stalk has a great deal of prognostic significance since patients with agenesis of the pituitary stalk run a greater risk of developing MPHD than those who show the vascular residue of the stalk. Patients in whom the pituitary stalk is not identifiable after Gd-DTPA have, in fact, a risk of developing additional pituitary hormone deficiencies evolving to panhypopituitarism that is 27 times greater than those with a residual vascular pituitary stalk [26]. The identification of EPP itself is helpful in the diagnosis and prognosis of patients with GH deficiency and the size of EPP may vary considerably between patients, ranging from small EPP to large EPP and even huge EPP (fig. 3, 5). In particular, the size and the location of EPP are early markers of evolving pituitary hormone deficiencies as reported in studies showing that small EPP surface area and/or hypothalamic-sited EPP

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are predictive of the development of MPHD [25, 27–30]. On the other hand, it is worth pointing out that a hypothalamic location of EPP is generally associated with complete pituitary stalk agenesis [27, 30] and that a huge EPP has been misdiagnosed as a subhypothalamic tumor in a patient subsequently submitted to unnecessary neurosurgery [31]. In a recent national survey of 3,000 French patients with nonacquired GH deficiency, 3 patients with central precocious puberty were found to have EPP at MRI [32].

Implications of EPP after Adult Height Achievement

The relationship between pituitary MRI features and final outcome has also been studied, showing that EPP is a determinant parameter in the prediction of growth response and adult height [22]. In particular, adult height in patients who showed permanent isolated GH deficiency, EPP and spontaneous puberty was similar to that of patients showing MPHD, EPP and induced puberty [33]. A subgroup of patients with idiopathic GH deficiency of childhood-onset who presented with congenital structural hypothalamic-pituitary abnormalities confirms that GH-deficient patients – defined as those with GH response <5 μg/l and with anterior pituitary hypoplasia, pituitary stalk agenesis and EPP at the level of the median eminence – are clearly candidates for permanent GHD in adult life [34]. Those with less severe posterior pituitary features as evidenced on MRI scans, on the other hand, have either an uncertain diagnosis or a likelihood of normal GH response after stimulation tests [28, 30, 35]. Indeed, pituitary function should be assessed periodically in subjects with EPP and isolated GH deficiency or combined pituitary hormone deficiency, as they may develop additional pituitary hormone deficiencies [30]. In our recent study, ACTH deficiency characterized a subset of patients with idiopathic GH deficiency and EPP regardless of the position of EPP, showing that several of these patients might have undiagnosed subclinical ACTH deficiency [36].

Conclusions

Establishing endocrine and MRI phenotypes is extremely helpful in the identification and management of patients with hypopituitarism, both in terms of possible genetic counseling and for early diagnosis of evolving anterior pituitary hormone deficiencies. The first-line identification of EPP during childhood is predictive of anterior pituitary dysfunction. Ectopic posterior pituitary appears to be a distinct entity reflecting an early abnormality in posterior pituitary migration, the molecular mechanism of which has yet to be perfectly clarified. It is hoped that a better understanding of posterior pituitary development will provide additional candidate genes for the great majority of individuals with ectopic posterior pituitary in whom, currently, the etiology is unknown.

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References 1 Mehta A, Dattani MT: Developmental disorders of the hypothalamus and pituitary gland associated with congenital hypopituitarism. Best Pract Res Clin Endocrinol Metab 2008;22:191–206. 2 Maghnie M, Di Iorgi N, Cesari S, Bernasconi S: Genetic forms of central diabetes insipidus; in Deal C (ed): MRI in Congenital Hypopituitarism: A Reference Guide. London, Rimedica Medical Education and Publishing, 2007, pp 99–112. 3 Asbreuk CH, van Doorninck JH, Mansouri A, Smidt MP, Burbach JP: Neurohypophysial dysmorphogenesis in mice lacking the homeobox gene Uncx4.1. J Mol Endocrinol 2006;36:65–71. 4 Xu C, Fan CM: Allocation of paraventricular and supraoptic neurons requires Sim1 function: a role for a Sim1 downstream gene PlexinC1. Mol Endocrinol 2007;21:1234–45. 5 Mitchell LA, Thomas PQ, Zacharin MR, Scheffer IE: Ectopic posterior pituitary lobe and periventricular heterotopia: cerebral malformations with the same underlying mechanism? AJNR Am J Neuroradiol 2002;23:1475–1481. 6 Kita A, Imayoshi I, Hojo M, Kitagawa M, Kokubu H, Ohsawa R, Ohtsuka T, Kageyama R, Hashimoto N: Hes1 and Hes5 control the progenitor pool, intermediate lobe specification, and posterior lobe formation in the pituitary development. Mol Endocrinol 2007;21:1458–1466. 7 Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M, Guillemot F, Kageyama R: Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development 2004;131:5539–5550. 8 Maghnie M, Di Iorgi N, Cesari S, Bernasconi S: Pituitary dysgenesis; in Deal C (ed): MRI in Congenital Hypopituitarism: A Reference Guide. London, Rimedica Medical Education and Publishing, 2007, pp 81–111. 9 Marcucio RS, Cordero DR, Hu D, Helms JA: Molecular interactions coordinating the development of the forebrain and face. Dev Biol 2005; 284: 48–61. 10 Kriangkrai R, Chareonvit S, Yahagi K, Fujiwara M, Eto K, Iseki S: Study of Pax6 mutant rat revealed the association between upper incisor formation and midface formation. Dev Dyn 2006;235:2134–2143. 11 de Graaff LC, Baan J, Govaerts LC, Hokken-Koelega AC: Facial and pituitary morphology are related in Dutch patients with GH deficiency. Clin Endocrinol (Oxf) 2008;69:112–116. 12 Larizza D, Maghnie M: Poland’s syndrome associated with growth hormone deficiency. J Med Genet 1990;27:53–55.

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13 Parano E, Trifiletti RR, Barone R, Pavone V, Pavone P: Arthrogryposis multiplex congenita and pituitary ectopia: a case report. Neuropediatrics 2000;31:325– 327. 14 Dupuis-Girod S, Gluckman E, Souberbielle JC, Brauner R: Growth hormone deficiency caused by pituitary stalk interruption in Fanconi’s anemia. J Pediatr 2001;138:129–133. 15 Oguz KK, Ozgen B, Erdem Z: Cranial midline abnormalities in Dubowitz syndrome: MR imaging findings. Eur Radiol 2003;13:1056–1057. 16 Baş F, Darendeliler F, Yapici Z, Gökalp S, Bundak R, Saka N, Günöz H: Worster-Drought syndrome (congenital bilateral perisylvian syndrome) with posterior pituitary ectopia, pituitary hypoplasia, empty sella and panhypopituitarism: a patient report. J Pediatr Endocrinol Metab 2006;19:535– 540. 17 Halász Z, Bertalan R, Toke J, Patócs A, Tóth M, Fekete G, Gláz E, Rácz K: Laterality disturbance and hypopituitarism. a case report of co-existing situs inversus totalis and combined pituitary hormone deficiency. J Endocrinol Invest 2008;31:74–78. 18 Sobrier ML, Maghnie M, Vie-Luton MP, Secco A, di Iorgi N, Lorini R, Amselem S: Novel HESX1 mutations associated with a life-threatening neonatal phenotype, pituitary aplasia, but normally located posterior pituitary and no optic nerve abnormalities. J Clin Endocrinol Metab 2006;91:4528–4536. 19 Machinis K, Pantel J, Netchine I, Léger J, Camand OJ, Sobrier ML, Dastot-Le Moal F, Duquesnoy P, Abitbol M, Czernichow P, Amselem S: Syndromic short stature in patients with a germline mutation in the LIM homeobox LHX4. Am J Hum Genet 2001; 69:961–968. 20 Machinis K, Amselem S: Functional relationship between LHX4 and POU1F1 in light of the LHX4 mutation identified in patients with pituitary defects. J Clin Endocrinol Metab 2005;90:5456– 5462. 21 Pfaeffle RW, Hunter CS, Savage JJ, Duran-Prado M, Mullen RD, Neeb ZP, Eiholzer U, Hesse V, Haddad NG, Stobbe HM, Blum WF, Weigel JF, Rhodes SJ: Three novel missense mutations within the LHX4 gene are associated with variable pituitary hormone deficiencies. J Clin Endocrinol Metab 2008;93:1062– 1071. 22 Maghnie M, Ghirardello S, Genovese E: Magnetic resonance imaging of the hypothalamus-pituitary unit in children suspected of hypopituitarism: who, how and when to investigate. J Endocrinol Invest 2004;27:496–509.

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23 Maghnie M, Rossi A, Di Iorgi N, Gastaldi R, TortoriDonati P, Lorini R: Hypothalamic-pituitary magnetic resonance imaging in growth hormone deficiency. Exp Rev Endocrinol Metab 2006;1:1–11. 24 Maghnie M, larizza D, Triulzi F, Sampaolo P, Scotti G, Severi F: Hypopituitarism and stalk agenesis: A congenital syndrome worsened by breech delivery. Horm Res 1991;35:104–108. 25 Maghnie M, Triulzi F, Larizza D, Preti P, Priora C, Scotti G, Severi F: Hypothalamic-pituitary dysfunction in growth hormone-deficient patients with pituitary abnormalities. J Clin Endocrinol Metab 1991;72:79–83. 26 Maghnie M, Genovese E, Villa A, Spagnolo L, Campan R, Severi F. Dynamic MRI in the congenital agenesis of the neural pituitary stalk syndrome: the role of the vascular pituitary stalk in predicting residual anterior pituitary function. Clin Endocrinol (Oxf) 1996;45:281–290. 27 Maghnie M, Strigazzi C, Tinelli C, Autelli M, Cisternino M, Loche S, Severi F: Growth hormone (GH) deficiency (GHD) of childhood onset: reassessment of GH status and evaluation of the predictive criteria for permanent GHD in young adults. J Clin Endocrinol Metab 1999;84:1324–1328. 28 Léger J, Danner S, Simon D, Garel C, Czernichow P: Do all patients with childhood-onset growth hormone deficiency (GHD) and ectopic neurohypophysis have persistent GHD in adulthood? J Clin Endocrinol Metab 2005;90:650–656. 29 Murray PG, Hague C, Fafoula O, Patel L, Raabe AL, Cusick C, Hall CM, Wright NB, Amin R, Clayton PE: Associations with multiple pituitary hormone deficiency in patients with an ectopic posterior pituitary gland. Clin Endocrinol (Oxf) 2008;69:597602. 30 di Iorgi N, Secco A, Napoli F, Tinelli C, Calcagno A, Fratangeli N, Ambrosini L, Rossi A, Lorini R, Maghnie M: Deterioration of growth hormone (GH) response and anterior pituitary function in young adults with childhood-onset GH deficiency and ectopic posterior pituitary: a two-year prospective follow-up study. J Clin Endocrinol Metab 2007; 92:3875–3884.

31 Werder EA, Zachmann M, Wichmann W, Valavanis A: Neurohypophyseal ectopy in growth hormone insufficiency. Horm Res 1989;31:210–212. 32 Ladjouze A, Soskin S, Garel C, Jullien M, NaudSaudreau C, Pinto G, Czernichow P, Léger J: GH deficiency with central precocious puberty: a new rare disorder associated with a developmental defect of the hypothalamic-pituitary area. Eur J Endocrinol 2007;156:463–469. 33 Maghnie M, Ambrosini L, Cappa M, Pozzobon G, Ghizzoni L, Ubertini MG, di Iorgi N, Tinelli C, Pilia S, Chiumello G, Lorini R, Loche S: Adult height in patients with permanent growth hormone deficiency with and without multiple pituitary hormone deficiencies. J Clin Endocrinol Metab 2006;91:2900– 2995. 34 Maghnie M, Aimaretti G, Bellone S, Bona G, Bellone J, Baldelli R, de Sanctis C, Gargantini L, Gastaldi R, Ghizzoni L, Secco A, Tinelli C, Ghigo E: Diagnosis of GH deficiency in the transition period: accuracy of insulin tolerance test and insulin-like growth factor-I measurement. Eur J Endocrinol 2005;152: 589–596. 35 Radetti G, di Iorgi N, Paganini C, Gastaldi R, Napoli F, Lorini R, Maghnie M: The advantage of measuring spontaneous growth hormone (GH) secretion compared with the insulin tolerance test in the diagnosis of GH deficiency in young adults. Clin Endocrinol (Oxf) 2007;67:78–84. 36 Maghnie M, Uga E, Temporini F, Di Iorgi N, Secco A, Tinelli C, Papalia A, Casini MR, Loche S: Evaluation of adrenal function in patients with growth hormone deficiency and hypothalamicpituitary disorders: comparison between insulininduced hypoglycemia, low-dose ACTH, standard ACTH and CRH stimulation tests. Eur J Endocrinol 2005;152:735–741.

Mohamad Maghnie, MD, PhD Department of Pediatrics, IRCCS G. Gaslini University of Genova, Largo Gerolamo Gaslini, 5 IT–16147 Genova (Italy) Tel. +39 010 563 6574, Fax +39 010 553 8265, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 95–113

Hyperinsulinism in Developmental Syndromes Ritika R. Kapoor ⭈ Chela James ⭈ Khalid Hussain London Centre for Paediatric Endocrinology and Metabolism, Great Ormond Street Hospital for Children, NHS Trust, and Developmental Endocrinology Research Group, Molecular Genetics Unit, Institute of Child Health, University College London, London, UK

Abstract Hyperinsulinism is a cause of recurrent and severe hypoglycaemia in the newborn and infancy period. Several developmental genetic syndromes are associated with hyperinsulinism. The underlying molecular mechanisms that lead to hyperinsulinaemic hypoglycaemia in most of these syndromes are unclear. Beckwith-Wiedemann syndrome (BWS) is the most common syndrome associated with hyperinsulinism. The incidence of hyperinsulinism in children with BWS is about 50%. The hyperinsulinaemic hypoglycaemia can be transient, which, in the majority of infants, will be asymptomatic and resolve within the first few days of life. Rarely patients with BWS may require a pancreatectomy. Other overgrowth syndromes such as Soto’s syndrome may overlap with BWS and present with hyperinsulinism. Patients with other rare syndromes such as Costello, Timothy and Kabuki syndromes can present with hyperinsulinaemic hypoglycaemia but the genetic mechanism(s) that leads to dysregulated insulin secretion in these syndromes is(are) still unclear. The congenital disorders of glycosylation (CDG) are a rapidly expanding group of metabolic syndromes with a wide symptomatology and severity. They all stem from deficient N-glycosylation of proteins. Hyperinsulinism has been described in congenital disorders of glycosylation, mostly in CDG-Ib but also as the leading symptom in a CDG-Ia patient. In summary, hyperinsulinism may be associated with a large number of developmental syndromes however the underlying molecular mechanisms that Copyright © 2009 S. Karger AG, Basel cause hyperinsulinism in these syndromes are still unknown.

Hyperinsulinism is characterised by dysregulated insulin secretion from the pancreatic β-cell leading to persistent hypoglycaemia. Hyperinsulinaemic hypoglycaemia is a major cause of mental retardation and brain injury in the childhood period [1]. The condition typically presents with severe hypoglycaemia in the newborn period but can also present in infancy and childhood period. There is marked heterogeneity in the clinical presentation and molecular biology of hyperinsulinaemic hypoglycaemia [2]. Nonsyndromic forms of hyperinsulinaemic hypoglycaemia may be transient or persistent. Transient forms of hyperinsulinaemic hypoglycaemia occur in association

Table 1. Summary of the developmental syndromes associated with hyperinsulinaemic hypoglycaemia Developmental syndromes

References

Pre- and post-natal overgrowth syndromes Beckwith-Wiedemann syndrome Sotos syndrome Simpson-Golabi-Behmel syndrome

[27–32] [50] [61]

Post-natal growth failure syndromes Kabuki syndrome Costellos syndrome

[76–78] [8, 69]

Chromosomal abnormality syndromes Trisomy 13 (Patau syndrome) Mosaic Turner syndrome

[81–84] [10]

Syndromes leading to abnormalities in calcium homeostasis (intermittent hyperinsulinaemic hypoglycaemia) Timothy syndrome

[93]

Contiguous gene deletion affecting the ABCC8 gene Usher Syndrome

[99, 100]

Congenital disorders of glycosylation syndromes Congenital disorder of glycosylation 1a Congenital disorder of glycosylation 1b Congenital disorder of glycosylation 1d

[101] [102, 103] [104]

with maternal diabetes mellitus (both insulin dependent and gestational), in infants born with intrauterine growth retardation (IUGR) and in infants who have suffered perinatal asphyxia. The commonest genetic cause of persistent hyperinsulinaemic hypoglycaemia are autosomal recessive mutations in the genes ABCC8 and KCNJ11 (encoding the two subunits SUR1 and KIR6.2, respectively) of the pancreatic β cell ATP-sensitive potassium channel (KATP) [3, 4]. Both transient and persistent hyperinsulinaemic hypoglycaemia may also be associated with a large number of developmental syndromes. Some of these developmental syndromes (such as Beckwith-Wiedemann syndrome; BWS) are relatively well defined phenotypically and genetically yet others (such as Kabuki syndrome) are not so clearly defined [5–7]. Overgrowth (both pre- and post-natal) may be a feature of some of these developmental syndromes causing hyperinsulinaemic hypoglycaemia. Intriguingly, some post-natal growth failure syndromes (such as Costello syndrome) have been reported in association with hyperinsulinaemic hypoglycaemia [8]. Several chromosomal syndromes have also been described in association with hyperinsulinaemic hypoglycaemia [9, 10]. Congenital defects of glycosylation (CDG), formerly called carbohydrate-deficient glycoprotein syndromes (CDGS), are a group of heterogeneous genetic diseases caused by deficient glycosylation of glycoconjugates,

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such as glycoproteins and glycolipids [11]. Hyperinsulinaemic hypoglycaemia may be a presenting feature of some of the CDG syndromes [12]. The underlying molecular mechanisms that cause hyperinsulinaemic hypoglycaemia in most of these developmental syndromes are currently unclear. This chapter provides an overview of the developmental syndromes that have been reported in association with hyperinsulinaemic hypoglycaemia. The focus of the review will be to give a brief clinical overview of each syndrome and then discuss the possible mechanisms leading to hyperinsulinaemic hypoglycaemia in these developmental syndromes. Table 1 summarises the developmental syndromes that have been reported in association with hyperinsulinaemic hypoglycaemia.

Hyperinsulinaemic Hypoglycaemia due to Pre- and Post-Natal Overgrowth Syndromes

Several pre- and post-natal overgrowth syndromes have been reported in association with hyperinsulinaemic hypoglycaemia. These include BWS, Sotos syndrome and Simpson-Golabi-Behmel syndrome. The most common overgrowth syndrome associated with hyperinsulinaemic hypoglycaemia is BWS. There is considerable overlap between these overgrowth syndromes, especially BWS and the SimpsonGolab-Behmel syndrome.

Beckwith-Wiedemann Syndrome BWS is a congenital overgrowth syndrome which is clinically and genetically heterogeneous. Phenotypically, BWS is associated with pre- and/or post-natal overgrowth, macroglossia, anterior abdominal wall defects, organomegaly, hemihypertrophy, ear lobe creases and helical pits and renal tract abnormalities. In one large study [13], the most frequent complications were macroglossia (97%), anterior abdominal wall defects (80%) and birth weight or post-natal growth >90th centile (88%). In this group of patients, hypoglycaemia was present in 63%. Other common clinical features in these patients included ear creases/pits, facial naevus flammeus and nephromegaly. Rarer complications included hemihypertrophy, moderate-to-severe developmental delay, congenital heart defects, polydactyly, neoplasia and cleft palate. The molecular aetiology of BWS is complex, involving alteration of the expression of multiple growth regulatory genes on chromosome 11p15 that are subject to genomic imprinting [14]. BWS is associated with a cluster of imprinted genes at human chromosome band 11p15.5 [15]. These genes are regulated by two independent imprinting centres called 1 and 2 with these imprinting centres regulating the expression of neighbouring genes [16]. Approximately 20% of patients with BWS have paternal uniparental disomy (UPD) for chromosome 11p15. Although the region

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of UPD varies, UPD for chromosome band 11p15 always involves both imprinting centres 1 and 2 [17]. UPD for chromosome 11 seems to be preferentially associated with specific features of BWS including hemihypertrophy, Wilms’ tumour, and hepatoblastoma [18]. UPD occurs in BWS as a post-fertilization mitotic recombination event resulting in somatic mosaicism for paternal UPD [19]. Other identified causes of BWS include loss of methylation at maternal imprinting centre 2 in 50%, gain of methylation at maternal imprinting centre 1 in 2–7%, rearrangements of 11p15 chromosome (translocation/inversion or duplication) in <1% and in 10–15% of cases, the aetiology is unknown [20]. Imprinting centre 1 is located at the distal end of 11p15 and regulates the expression of two genes: insulin-like growth factor 2 (IGF2) and H19 [21]. The IGF2 and H19 genes are normally regulated so that only H19 is expressed on the maternal chromosome and only IGF2 is expressed on the paternal chromosome. Paternal isodisomy results in two active copies of the growth factor IGF2 and no expression of H19. Hypermethylation of H19 causes the same changes in gene expression resulting in the increased risk of hemihypertrophy and an increased risk of Wilms’ tumour [22]. Imprinting centre 2 is centromeric to imprinting centre 1 and contains several imprinted genes, two of which, to date, have been implicated in BWS. These include KCNQ1OT1 [23] and CDKN1C [24]. Normally, the maternal allele of imprinting centre 2 is methylated silencing the expression of KCNQ1OT1 so that KCNQ1OT1 is expressed only from the paternal chromosome. Loss of maternal methylation at imprinting centre 2 and concomitant biallelic expression of KCNQ1OT1 is seen in 50% of patients with sporadic BWS [22]. Imprinting centre 2 also regulates cyclindependent kinase inhibitor 1C (CDKN1C) so that normally preferential maternal expression occurs. Loss of maternal methylation at imprinting centre 2 is associated with decreased CDKN1C expression [25]. Cyclin-dependent kinase inhibitor 1C and its protein product, p57KIP2, is a member of the cell cycle regulatory proteins important for controlling growth. Paternal UPD 11 results in biallelic expression of KCNQ1OT1 and reduced expression of CDKN1C in the isodisomic cell population. The incidence of hyperinsulinaemic hypoglycaemia in children with BWS is about 50% [26]. This hypoglycaemia can be transient which in the majority of infants will be asymptomatic and resolve within the first few days of life. In about 5% of children the hyperinsulinaemic hypoglycaemia can be persistent and extend beyond the neonatal period requiring either continuous feeding, medical therapy or, in rare cases, partial pancreatectomy [27–32]. In this group of children, the hypoglycaemia can be severe causing significant brain damage and may be a factor contributing to sudden death [33]. The underlying mechanism(s) leading to persistent hyperinsulinaemic hypoglycaemia in this syndrome is(are) unclear. Recent advances in understanding the molecular basis of congenital hyperinsulinism may give some insights into the mechanisms of hyperinsulinaemic hypoglycaemia in patients with BWS. The commonest cause of congenital hyperinsulinism are mutations in the ABCC8 and KCNJ11 genes that encode for the protein components

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SUR1 and Kir6.2 of the KATP channels in the pancreatic β cell [3, 4]. Histologically, congenital hyperinsulinism can be divided into diffuse, focal and atypical forms. The diffuse form is due to autosomal-recessive mutations in the ABCC8 and KCNJ11 genes (located on chromosome 11p15) affecting all of the pancreatic β cells. Focal hyperinsulinism results from a paternally inherited KATP channel mutation together with somatic loss of the maternal chromosome 11p15 region [34, 35] in a limited area of the pancreas causing adenomatous hyperplasia in the area. Maternal loss of heterozygosity (LOH) in this region involves the loss of maternally expressed tumour suppressor genes (H19 and CDKN1C) and duplication of the paternally expressed IGF2 (insulin-like growth factor 2) gene which is believed to play a major role in foetal growth. It is thought that it is this imbalance of the imprinted genes that leads to focal hyperplasia of the cells (due to overexpression of IGF2) along with reduction to homozygosity of the KATP channel mutation that leads to insulin hypersecretion within the focal lesion. In addition, atypical diffuse forms are also described due to mosaic KATP channel mutations in the pancreas [36]. Paternal UPD11 or H19 hypermethylation resulting in overexpression of IGF2 is a plausible explanation for the hyperinsulinism observed in patients with BWS. In theory, this overexpression of IGF2 could lead to hyperplasia of the pancreatic β-cells through its growth-promoting effects [34]. However, there is no hyperinsulinaemic hypoglycaemia in a transactivation IGF2 mouse model of BWS [37] and no significantly increased risk of hyperinsulinaemic hypoglycaemia in patients with increased IGF2 expression [38]. Patients with BWS due to paternal UPD11 have an increased risk of hypoglycaemia compared to other genetic abnormalities [13]. However, in these patients there is no evidence for duplication of INS, HRAS1 and IGF2 [39] genes or overexpression of the INS and IGF2 genes [40]. Hence, overexpression of IGF2 cannot be the only mechanism causing hyperinsulinaemic hypoglycaemia in children with BWS and other mechanism(s) must exist. Understanding the pancreatic histology in BWS is limited to the severe cases in which the patient has died or had partial pancreatectomy [28–32]. The histological findings in these patients are one of pancreatic β cell hyperplasia and hypertrophy. These findings are similar to those in patients with the diffuse form of congenital hyperinsulinism [40]. A reduction in somatostatin-producing cells has been noted [41] but the significance of this observation is unclear. Several patients with BWS have now been reported where in vitro electrophysiological properties of the KATP channels have been studied following pancreatectomy. In 1 patient [31], the hyperinsulinaemic hypoglycaemia was severe and unresponsive to medical therapy with diazoxide. This patient required a near total pancreatectomy and the BWS was due to mosaic paternal UPD for chromosome 11p15. The electrophysiological properties of the KATP channels in pancreatic β cells demonstrated abnormal function of the potassium channels [31]. A similar patient has been reported by Giurgea et al. [32] where a clear defect in the function of the KATP channels was demonstrated in vitro but again no mutations were found in the KATP

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channel genes. The mechanism(s) by which mosaic paternal uniparental disomy for 11p15 causes a chanellopathy is yet to be understood. However, this would account for <5% of hyperinsulinaemic hypoglycaemia in cases of BWS. The vast majority of cases will respond to medical therapy with diazoxide.

Sotos Syndrome Sotos syndrome was first described by Sotos et al. [43] in 5 children with a disorder characterized by excessively rapid growth, acromegalic features and mental retardation. It is now established that the three cardinal features present in >90% of the cases of Sotos syndrome include a characteristic facial appearance, overgrowth and learning disability [44, 45]. The facial appearance is most notable in early childhood and includes macrodolicocephaly, pointed chin, downslanting palpebral fissures, malar flushing and a receding hairline. Facial dysmorphism may also include prominent jaw, high arched palate, large ears, anteverted nostrils and micrognathia [46]. Intellectual impairment is a common feature (97%) although the degree of learning disability is extremely variable. Overgrowth in Sotos is particularly pronounced in the first year of life and in early childhood. However, final height is within the high normal range in the majority [47]. Among the designated major features, an advanced bone age is the most common feature and occurs in 75% of affected children. The abnormalities on cranial imaging are nonspecific with ventricular dilatation being the commonly reported feature. Vesico-ureteric reflux is the most common renal anomaly, though structural abnormalities (duplex/ absent kidney, urethral stenosis and pelviureteric junction obstruction) have also been described. Similarly, cardiac anomalies, seizures and scoliosis of various types and severity have been recognized in association with Sotos syndrome. Many other clinical features have been reported in individuals with Sotos syndrome including neonatal hypoglycaemia [44, 48, 49]. However, little is known about the cause of hypoglycaemia in this condition. Hyperinsulinaemic hypoglycaemia has been described in one patient with a molecular diagnosis of Sotos syndrome which was persistent at 5 years of age [50]. Mutations in the NSD1 (nuclear receptor set domain containing protein 1) gene are identifiable in the majority of patients (>90%) with Sotos syndrome [44, 51–55]. NSD1 gene is located at chromosome 5q35 and its exact function is not known. It is thought that it codes for a protein that functions as a histone methyl-transferase [56]. It is possible that its complex role in histone methylation may play a role in establishing imprinting of the 11p15 region [50]. As discussed above, BWS occurs due to dysregulation of the imprinted growth regulatory genes in this region of chromosome 11p15. It is tempting to speculate that the phenotypic resemblance, including hyperinsulinaemic hypoglycaemia in the two syndromes may be due to the effect of NSD1 mutations on the imprinted genes in this region [50].

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Simpson-Golabi-Behmel Syndrome Simpson-Golabi-Behmel Syndrome (SGBS) is a rare and complex overgrowth syndrome characterized by macrosomia, macroglossia, skeletal abnormalities, extramammary nipple, and genitourinary abnormalities. There is considerable overlap with other overgrowth syndromes, especially BWS. The diagnosis of SGBS is usually clinical and the typical coarse facial appearance with greater likelihood of skeletal and vertebral abnormalities (polydactyly, syndactyly, scoliosis, pectus excavatum and rib malformations) distinguishes it from other overgrowth syndromes. The facial appearance described includes low-set posteriorly rotated ears with abnormal helix, pits or tags, epicanthic folds, down-slanting palpebral fissures, broad and upturned nose, protruding jaw, high arched or cleft palate with notched alveolar ridge, macroglossia or a deep groove in the middle of the tongue, and short or webbed neck [57–59]. Hypoglycaemia has been reported in SGBS syndrome [60] but data on the prevalence and severity of hypoglycaemia is lacking. It is believed to be related to hyperinsulinism and islet cell hyperplasia [61]. SGBS is associated with loss of function mutations of the X-linked GPC3 gene encoding glypican-3, a cell-surface heparan sulfate proteoglycan [62]. The function of GPC3 in somatic growth remains unknown. It has been speculated that GPC3 downregulates IGF2 [62, 63] and this could account for the phenotypic resemblance between BWS and SGBS [64]. However, data from studies by Eric Chiao et al. [65] comparing growth patterns of double mutants lacking GPC3 and IGFs did not support the hypothesis. Nevertheless, their studies do support the possibility that IGF2 and GPC3 could act via independent signalling pathways that converge on common downstream effectors. Further understanding of the action of GPC3 and study of its interaction with growth factors will shed light on the mechanism of somatic overgrowth seen in this condition.

Hyperinsulinaemic Hypoglycaemia in Post-Natal Growth Failure Syndromes

Hyperinsulinaemia hypoglycaemia may be a feature of some post-natal growth failure syndromes such as observed in Costello and Kabuki syndromes. Both of these syndromes cause post-natal growth failure.

Costello Syndrome Costello syndrome is a rare congenital disorder affecting multiple organ systems, encompassing severe failure to thrive, cardiac anomalies including hypertrophic cardiomyopathy and atrial tachycardia, tumour predisposition, and cognitive impairment. [66]. Costello syndrome shares findings with cardio-facio-cutaneous syndrome (CFC) caused by a mutation in the KRAS gene and Noonan syndrome [67]. Recently,

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it was shown that Costello syndrome is caused by heterozygous de-novo point mutations in HRAS gene which lies downstream of SHP2 the RAS-MAPK (mitogen activated protein kinase) signalling cascade [68]. De novo heterozygous missense mutations in HRAS codons 12 and 13 disturb the intrinsic GTP hydrolysis. Hyperinsulinaemic hypoglycaemia has been observed in patients with Costello patients [8, 69]. Histological examination of the pancreas in these patients at postmortem showed a grossly normal pancreas but microscopically there was an increased number of islets of Langerhans (islet cell hyperplasia) distributed equally in the head and tail. The underlying mechanisms leading to hyperinsulinaemic hypoglycaemia in Costello syndrome are unclear at present but may be related to the marked islet cell hyperplasia. Mutations at codons 12 and 13 of the HRAS gene are in constitutively active GTPbound conformation and activate downstream effectors such as MAPK, PI-3 kinase and RalGDS [70]. Cell proliferation studies from patients with Costello syndrome show increased uptake of 5-bromodeoxyuridine as compared with controls [68]. This suggests the effect of an increased growth factor dependent proliferation, which might partially explain the mechanism of organ hypertrophy (and possibly the pancreatic hyperplasia) in Costello syndrome.

Kabuki Syndrome Kabuki syndrome is a multiple malformation/mental retardation syndrome characterized by facial dysmorphism, skeletal anomalies, dermatoglyphic abnormalities, short stature, and mental retardation. The diagnosis is usually based on the characteristic facial appearance which includes long palpebral fissures with eversion of the lateral portion of the lower eyelids; broad, arched eyebrows with lateral sparseness; short nasal columella with depressed nasal tip and large, prominent or cupped ears [71]. Various other manifestations involving other systems (cardiovascular, renal, endocrine, immunological, ophthalmological, neurological and gastrointestinal) are also known to be associated with this clinically heterogeneous syndrome. The genetic basis of Kabuki syndrome is unknown. Most cases of Kabuki syndrome are sporadic; however, vertical transmission in some familial cases suggest an autosomal-dominant inheritance. Several isolated cytogenetic anomalies have been reported in individual cases of KS that are not thought to be causally related to KS. Recently, Milunsky and Huang [72] reported 6 unrelated patients with KS and an 8p22–8p23.1 duplication and suggested this as a cause of KS. However, this observation was not confirmed by other groups [73–75] and it may be that the patients studied by Milunsky and Huang [72] had atypical KS or possibly another syndrome with features similar to KS [75]. Hyperinsulinaemic hypoglycaemia has been reported in various case reports of patients with KS [76–78]. More recently, Genevieve described congenital hyperinsulinism

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in 4 cases of Kabuki syndrome [78]. All 4 responded to diazoxide therapy and were negative for a KATP channel mutation. Advances in molecular genetics and understanding of the genetic aetiology of this interesting condition in future will shed light on the cause of the varied manifestations seen in KS, including hyperinsulinism. However, it is important to recognise that hyperinsulinism is a treatable cause of hypoglycaemia in this condition where children are already prone to seizure disorder and mental retardation.

Hyperinsulinaemic Hypoglycaemia in Chromosomal Abnormality Syndromes

Hyperinsulinaemic hypoglycaemia has been observed in two syndromes associated with chromosomal abnormalities. These include Patau syndrome and Mosaic Turner syndrome.

Trisomy 13 (Patau Syndrome) Trisomy 13 is the third most common trisomic disorder in human live births. The prevalence is about 1 in 5,000 births with high infant mortality. Common phenotypic abnormalities include severe mental retardation, cleft lip and/or palate, hypotonia, skeletal abnormalities and heart defects [79, 80]. In the literature, 4 cases of trisomy 13 have been associated with hyperinsulinaemic hypoglycaemia [81–84]. The pathogenesis of hyperinsulinaemic hypoglycaemia in trisomy 13 remains unknown. Overdosage of certain gene(s) may lead to hyperinsulinism. Such candidate genes include the insulin promoter factor-1 gene (IPF1) located at 13q12.2, which binds the specific sequence within the promoter region of the insulin gene and activates its transcription [85]. The other possible overdosage gene is the caudal-type homeobox transcription factor 2 gene (CDX2) located at 13q12.3, encoding a homeodomain protein that binds an A/T-rich sequence in the insulin promoter and stimulates its transcription [86]. Ductuloinsular complexes (incorporation of ducts into islets of Langerhans or the close association of endocrine cells with small knots of ductules) have been described in the pancreatic pathology in some patients with trisomy 13, which was interpreted as nesidioblastosis [87, 88]. Nesidioblastosis describes the budding of pancreatic islets from the ducts and is not pathogenic for congenital hyperinsulinism.

Mosaic Turner Turner syndrome is a common genetic cause of short stature in females. The characteristic clinical stigmata are variable, but include growth retardation with reduced adult height, gonadal insufficiency, and infertility with or without additional phenotypical features such as webbing of the neck and lymphoedema. Turner syndrome is

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typically associated with impaired glucose tolerance. The underlying pathophysiological mechanism/s of impaired glucose tolerance in girls with Turner syndrome is unclear. Some studies suggest that insulin resistance seems to be a very early metabolic defect that may be restricted to nonoxidative pathways of intracellular glucose metabolism [89]. Yet other studies suggest that the increased prevalence of impaired glucose tolerance in patients with Turner syndrome may be due to a primary defect in the pancreatic β cells to produce insulin and this may be related to haploinsufficiency for unknown X-chromosome genes in this metabolic phenotype [90, 91]. The gene for insulin receptor substrate (IRS) 4 has been mapped to Xp22.3–23 and is a putative candidate gene for an insulin-resistant phenotype [92], but its role in regulating insulin secretion from pancreatic β cells is not known. We described the first case of a child with a complex mosaic Turner genotype and hyperinsulinaemic hypoglycaemia responsive to diazoxide therapy [10]. Cytogenetic analysis in this patient showed four cell lines; one with 45X, the others with an additional small ring chromosome, a small marker chromosome, and both the ring and marker chromosomes, respectively. FISH studies showed the abnormal chromosomes to originate from an X. The X inactivation locus (XIST) was present in the ring, but not in the marker chromosome. The pathogenesis of the persistent hyperinsulinaemic hypoglycaemia in our mosaic Turner syndrome patient remains unknown. It is interesting to note that monosomy X increases the risk of glucose intolerance and subsequent diabetes mellitus. This suggests that haploinsufficiency of unknown gene(s) on the X-chromosome may be one mechanism by which these patients have impaired glucose tolerance. In our patient, three cell lines contained additional X chromosome material, the marker X without the XIST locus and therefore not inactivated. In contrast to the (pre-) diabetic Turner syndrome, our patient may have an over dosage of certain gene(s) due to non-inactivated additional X chromosome material. Alternatively, pancreatic β cells may lack normal X chromosome gene(s) in cell lines with activated ring X and inactivated normal X.

Hyperinsulinaemic Hypoglycaemia due to Syndromes Leading to Abnormalities in Calcium Homeostasis

Alterations in the intracellular calcium concentration play a pivotal role in regulating insulin secretion from pancreatic β cells. Intermittent hyperinsulinaemic hypoglycaemia has been reported in patients with Timothy syndrome.

Timothy Syndrome Timothy syndrome is characterized by multiorgan dysfunction, including lethal arrhythmias, webbing of fingers and toes, congenital heart disease, immune deficiency,

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intermittent hyperinsulinaemic hypoglycaemia, cognitive abnormalities, and autism [93]. The disorder results from a de novo glycine to arginine change in the 406th codon of the CACNA1C gene [93]. CACNA1C encodes the alpha-1 subunit of the L-type voltage dependent Ca2+ channel (L-VDCC) [94]. Functional expression in heterologous systems demonstrates that the disease-associated mutation causes abnormal Ca2+ current. This gain-of-function mechanism is mediated through failed channel inactivation. Previous studies have shown that the CaV1.2 gene is expressed in heart, brain, smooth muscle, pituitary and adrenal glands [95]. In pancreatic β-cells, the expression of mRNA has been demonstrated for many high-voltage-activated calcium (HVC) channels [96]. These include the α1A (Cav2.1), α1B (Cav2.2), α1C (Cav1.2), α1D (Cav1.3), and α1E (Cav2.3). Regardless of which HVA calcium channels are preferentially coupled to insulin secretion, maintenance of regulated calcium influx remains vital for normal β cell activity and survival. The HVA channel class may play an important role in the maintenance of calcium homeostasis in insulin-secreting cells in addition to providing localized calcium influx for vesicular secretion. In the pancreas, Ca2+ mediates insulin secretion by pancreatic β-cells. Episodic dysfunction of CaV1.2 signalling likely accounts for the intermittent hyperinsulinaemic hypoglycaemia observed in these patients.

Hyperinsulinaemic Hypoglycaemia due to Contiguous Gene Deletion Affecting the ABCC8 Gene

The ABCC8 gene encodes for the SUR1 component of the pancreatic β-cell KATP channel. Given the unique role of the SUR1 in regulating insulin secretion any genetic defect that deletes the ABCC8 gene will lead to hyperinsulinaemic hypoglycaemia. Usher syndrome and severe hyperinsulinaemic hypoglycaemia have been reported in several patients due to a contiguous gene deletion.

Usher Syndrome Usher syndrome is an autosomal-recessive condition and describes the combination of progressive pigment retinopathy resulting in blindness and congenital sensorineural hearing loss [97]. Clinically, Usher syndrome is a heterogeneous condition and is differentiated into three types based on the severity and progression of the hearing impairment and by the presence or absence of vestibular symptoms. Type I Usher syndrome is the commonest and most severe type of Usher syndrome with profound hearing loss and defective vestibular function. At least 7 different genes (types 1A-1G) are responsible for type I Usher syndrome [97, 98]. Severe hyperinsulinaemic hypoglycaemia has been described in three patients with Usher type IC [99, 100]. These patients also had features of renal tubular dysfunction

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and severe enteropathy. This combination of clinical features was caused by a 122-kb deletion of the short-arm of chromosome 11 encompassing more than half of the ABCC8 gene (causing the hyperinsulinism), and was instrumental in the identification of the gene causing type IC Usher syndrome (USH1C gene). The ABCC8 gene codes for the SUR1 protein on the KATP channels in the pancreatic β cells. Hence, it was not surprising for the deletion which removed 21 of the 39 exons of the ABCC8 gene to cause a chanellopathy leading to hyperinsulinism that was unresponsive to diazoxide. Two of these patients required a near total pancreatectomy and the third required total pancreatectomy to control the hypoglycemia. The histology of the pancreas was consistent with the diffuse form of the disease [100].The other clinical manifestations seen in these patients were explained by partial deletion of the USH1C gene that is expressed in the inner ear, photoreceptors of the eye, renal tubular epithelium and the gut [99].

Hyperinsulinaemic Hypoglycaemia due to Congenital Disorders of Glycosylation Syndromes

Congenital defects of glycosylation (CDG), formerly called carbohydrate deficient glycoprotein syndromes (CDGS), are genetic diseases caused by deficient glycosylation of glycoconjugates, such as glycoproteins and glycolipids [11]. The glycans on proteins are either N-linked (to the amide group of asparagine via an N-acetylglucosamine residue) or O-linked (to the hydroxyl group of serine or threonine via an N-acetylgalactosamine or a xylose residue). In humans, most known CDG are N-glycosylation defects; only a few defects are known in O-glycosylation, specifically in glycosaminoglycan synthesis. The clinical spectrum of the different types of CDG discovered so far is variable, ranging from severe multisystemic disorders to disorders restricted to specific organs. The CDG causing sialic acid deficiency of N-glycans can be diagnosed by isoelectrofocusing of serum sialotransferrins [11]. Hyperinsulinaemic hypoglycaemia has been reported in CDG syndromes 1a, 1b and 1d [101–104]. Hyperinsulinaemic hypoglycaemia may be the presenting symptom in patients with CDG type 1a (phosphomannomutase deficiency) without any of the other manifestations at the time of presentation [101]. Several patients with CDG type 1b have now been reported [102, 103]. Again hyperinsulinaemic hypoglycaemia was the main presenting symptom in some of these patients. These patients subsequently developed protein-losing enteropathy, liver disease, and coagulopathy; the typical clinical features of CDG. Patients with CDG type 1b were treated with diazoxide and oral mannose which corrected the blood glucose abnormality. Finally, 1 patient with CDG type 1d has been reported [104]. This patient again presented with severe hyperinsulinaemic hypoglycaemia in addition to other clinical features such as a Dandy-Walker malformation, facial dysmorphism, and profound hypotonia. Post-mortem examination in this patient revealed islet cell hyperplasia with increased β cell mass.

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The molecular basis for the hyperinsulinaemic hypoglycaemia in patients with CDG syndromes probably involves defects in glycosylation of the components of the KATP channels. Pancreatic β cell KATP channels mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability [105]. The number of plasma membrane KATP channels determines the sensitivity of β-cell to glucose stimulation. The KATP channel is formed in the endoplasmic reticulum (ER) on co assembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits [106]. SUR1 is present on the plasma membrane and is composed of the mature, complex glycosylated form. Mutagenesis studies have demonstrated the presence of two N-linked glycosylation sites on SUR1 at positions Asn10 and Asn1050 corresponding to the N terminus and the external loop following transmembrane segment 12 [107]. Glycosylation of SUR1 promotes surface expression of KATP channels with glycosylated mutants resulting in endoplasmic reticulum retention [108]. Hence reduction in the surface expression of KATP channels will lead to unregulated insulin secretion.

Conclusion

Hyperinsulinaemic hypoglycaemia is associated with a large number of developmental syndromes. The genetic basis of some of these developmental syndromes are beginning to be well understood (for example BWS) but others are still poorly defined (such as Kabuki syndrome). Understanding the molecular mechanisms of hyperinsulinaemic hypoglycaemia in these developmental syndromes will provide novel insights into insulin and glucose physiology. The recognition and appropriate management of hyperinsulinaemic hypoglycaemia in patients with developmental syndromes is important as these patients may already have an increased risk of mental retardation.

Acknowledgments This work was undertaken at GOSH/UCL Institute of Child Health which received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centre funding scheme.

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Dr. K. Hussain Developmental Endocrinology Research Group Molecular Genetics Unit, Institute of Child Health, University College London 30 Guilford Street, London WC1N 1EH (UK) Tel. +44 20 7 905 2128, Fax+44 20 7 404 6191, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 114–134

Developmental Syndromes: Growth Hormone Deficiency and Treatment Laura Mazzanti ⭈ Federica Tamburrino ⭈ Rosalba Bergamaschi ⭈ Emanuela Scarano ⭈ Francesca Montanari ⭈ Michele Torella ⭈ Elisa Ballarini ⭈ Alessandro Cicognani Department of Pediatrics, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy

Abstract Developmental syndromes are characterized by numerous phenotypical signs and malformations. In most of them such as Turner, Noonan, Prader-Willi, Silver-Russel, Williams, Kabuki, Leri-Weill syndrome and skeletal dysplasias, short stature is a common feature. Growth defect is very often related to a defect in cellular growth, but some unknown abnormality in GH action is possible. Recently, the greater availability of recombinant GH has expanded the interest towards GH secretion and therapy also in developmental syndromes. We recognize syndromes associated with GH deficiency (GHD), showing a developmental midline defect such as Pallister-Hall syndrome, septo-optic dysplasia, but many of these conditions do not have a convincing link with GHD. Moreover, some conditions, in particular the well-studied Turner syndrome, that do not have a real GHD, have proven to benefit from GH therapy at supra-physiological doses obtaining a higher final height than the expected one according to the natural history. This has expanded the indications for GH therapy. The aim of our paper is to review the literature on GH secretion, on the effects and costs-benefits of GH therapy in many dysmorphic syndromes, presenting some results of GH secretion and therapy obtained in our Copyright © 2009 S. Karger AG, Basel experience.

Short stature is a common feature of developmental syndromes. The dysmorphic syndromes are characterized by numerous phenotypical signs and malformations that interest different developmental areas and recognize an etiological cause known (cytogenetic anomalies, gene mutations, gene/environment interactions) or not known. In genetic syndromes growth defect is very often related to a deficit in cellular growth, caused by the genetic defect, but it is possible to hypothesize that it may determine some unknown abnormality in the action of growth hormone (GH). In recent years, the larger availability of recombinant GH has expanded the interest towards GH secretion and therapy also in developmental syndromes.

We recognize syndromes that are generally associated with GH deficiency (GHD), showing a developmental midline defect such as Pallister-Hall syndrome, septo-optic dysplasia and so on, but many of the dysmorphic conditions, apart from PraderWilli syndrome that has a proven disorder of GH secretion, do not have a convincing link with GHD. Moreover, some conditions, in particular the well-studied Turner syndrome, that do not have a real GHD have proven to benefit from this therapy at supra-physiological doses and this has expanded the indications for GH therapy. The aim of this paper is to review the literature on GH secretion on the effects and cost benefits of GH therapy in the most frequent genetic dysmorphic syndromes such as Turner, Noonan, Prader-Willi, Silver-Russel, Williams, Kabuki, Leri-Weill, skeletal dysplasias and in some that are less frequent. We will also present some data of the results of GH secretion and therapy obtained in our patients with these conditions.

Turner Syndrome

Etiology Turner syndrome (TS) is the most common sex-chromosome abnormality in females and affects approximately 1/1,500–2,500 liveborn females. It is due to complete or partial X-monosomy.

Clinical Signs Typical signs are short stature, dysmorphic features (lymphedema, webbing of the neck, cubitus valgus), gonadal dysgenesis, CHD, renal anomalies, infertility, normal intelligence with some cognitive difficulties in mathematic problems or visuospatial processing.

Growth Short stature is the most common finding. Growth retardation begins, very often, prenatally (although severe intrauterine growth retardation is uncommon) and persists through early childhood. Final height (FH) in TS patients is approximately 20 cm below the mean for the respective ethnic groups [1]. Growth failure is frequently subtle and cumulative, often resulting in delayed diagnosis. It is now clear that multiple causes are likely to be involved in short stature: chromosomal anomalies, specific genes (haploinsufficiency of SHOX, PHOG) and nongenetic factors such as lymphedema.

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Fig. 1. Final height (cm) of each subject with TS according GH therapy groups: untreated and treated with GH for less and more of 4 years. The line of 142.5 cm corresponds to the 50th centile for untreated Italian TS girls.

Growth Hormone Secretion and Therapy GHD has been reported in some patients with TS [2], but it has been shown to be of no importance in influencing growth before therapy, response to GH therapy and final height. Growth failure seems to be related to an impaired response to GH. GH therapy at supraphysiological doses enhances growth in children and adolescents with TS. It has been reported that GH therapy provides a good auxological result in the short term and improves final height, especially with high doses for long periods. An early start of GH therapy allows normalization of height in the prepubertal age and the introduction of estrogen therapy at a physiological age. An Italian multicenter study [3] reported a height gain of 1.5 ± 0.9 SD after 5 years of therapy in 29 patients (1.4–5.9 years). Recently, in a group of 41 patients treated at a very early age (9 months–4 years) Davenport et al. [4] reported a height gain of 1.6 SD after 2 years of therapy compared with an age-matched group and 93% of the patients showed a normalization of height before 6 years of age. The dose of GH generally recommended for use in TS is 0.375 mg/kg/week, double the dose used in GHD children. Different studies reported a height gain in FH with high doses of GH between 2.1 and 16.9 cm. The highest gain was observed by van Parenen et al. [5] with higher doses of GH [FH gain 15.7 ± 3.5 cm (GH dose 0.46 mg/kg/week) and 16.9 ± 5.2 cm (GH dose 0.61 mg/kg/week)]. The Canadian Randomized Controlled Trial is the only randomized study that showed a height gain of 7.3 ± 9.2 cm (95% Cl 5.3–9.2 cm; + 1.1 SDS) in treated patients (0.35 mg/kg/week) vs. an untreated group [6].

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Personal Data In our series, 133 patients reached FH: 25 of them without GH and 108 patients treated with GH (0.33 mg/kg/week) for a mean period of 6.2 ± 2.7 years (range: 2–12.8 years); 74 patients treated for a period >4 years, 54 patients for >6 years and 29 patients >8 years; 24 patients developed spontaneous menarche. FH were 148.6 ± 5.4 cm in patients treated for a period <4 years vs. 152.9 + 5.3 cm in patients treated >4 years; 154.2 + 4.6 cm >8 years of GH therapy (fig. 1). At multiple regression analysis, height velocity in the 1st year of therapy, pretherapy height, GH duration (p = 0.00001), age at the start of GH therapy (p = 0.008) and target height (TH) (p = 0.009) are the most important factors influencing FH. Spontaneous menarche seems to have a negative influence on FH (T = –1.9, p = 0.06). Our results confirm the importance of dosage, age at the start and duration of GH therapy, in particular a minimum of 4 years of therapy, to obtain a good result in FH in subjects with TS.

Noonan Syndrome

Noonan syndrome (NS) is a quite frequent genetic condition (1/1,000–2,500 live births).

Etiology An autosomal-dominant inheritance has been suggested. The PTPN11 gene has been identified as the disease gene, located on chromosome 12 (12q24.1), in about 50% of the NS patients. This gene encodes the protein tyrosine phosphatase SHP2, that is involved in postreceptor signaling of developmental processes in the RAS-MAPK (mitogen-activated protein kinase) pathway; the mutation produces a gain of function that impairs the GH post-receptorial signal. Recently, other genes have been identified in this pathway (KRAS, SOS1, RAF1).

Clinical Data The variable phenotype accounts for the difficulty in diagnosis, that remains mainly clinical, therefore many scoring systems have been prepared and the most practical was prepared by van der Burgt. The characteristic pattern of dysmorphic features includes the face, trunk and extremities, congenital heart defects such as pulmonary valvular stenosis and other cardiac abnormalities, cryptorchidism, bleeding diathesis.

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H-SDS increment vs baseline

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Fig. 2. Comparison of height gain SDS in patients with Noonan syndrome with and without PTPN11 mutation.

Growth Growth retardation is a consistent feature and most affected patients have a height below the 3rd centile with a FH of 162 cm for males and 153 cm for females.

Growth Hormone Secretion and Therapy GH secretion has been shown to be frequently abnormal, but classical GHD has not been found; however, it has been shown that some patients show alterations of the GH-IGF-1 axis (neurosecretory dysfunction). No correlation was found between GHD and growth response to therapy. In the literature, short-term studies on few patients showed that GH therapy was effective in increasing height velocity. Recently, in a Swedish study [7] on 25 prepubertal children treated with GH, 18 of them reached the FH, very close to the TH, with the gain of 1.7 SD on pretreatment height. In the study of Noordam and Otten [8], 38 patients treated with GH reached FH after a mean of 7.1 years of GH treatment. The positive response in the 1st year of treatment (about 1 SD) was maintained during the following years and even up to FH. Some recent studies have shown that the presence of PTPN11 mutation may influence height velocity and FH. In the study by Binder et al. [9], GH secretion and IGF-1/ IGFBP3 levels were compared in PTPN11-positive with PTPN11-negative patients; the levels of IGF-1 (–2.0 vs. –1.1 SD) and IGFBP3 (–0.9 vs. 0.4 SD) were found to be lower in the PTPN11+ group with GH levels higher during spontaneous secretion at night and on arginine stimulation. The authors suggest that PTPN11 mutations may cause a mild GH resistance by a postreceptor signaling defect, which may contribute

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to growth failure and the relatively poor response to GH in NS. Patients with NS with heart disease may be submitted to GH therapy and only the subjects with hypertrophic cardiomyopathy (HCM) have been excluded. In all NS subjects, before GH therapy and yearly during treatment, echocardiography should be recommended to exclude and monitor the development of HCM [10].

Personal Data In our group of 38 NS patients, studied for PTPN11, 16 children were treated with GH in a 10-year follow-up. 6 patients had the PTPN11 mutation. After the 1st year of GH therapy, there were no significant height differences between PTPN11– and PTPN11+ patients (–2.7 vs. –2.4 SDS ). After 5 years of therapy PTPN11– patients achieved a stature of –2.1 SDS and PTPN11+ remained at –2.3 SDS. After 10 years of therapy PTPN11– achieved a stature of -1.5 SDS and PTPN11+ –2.2 SDS (fig. 2). In conclusion, after 10 years of therapy, PTPN11– had a height gain vs. pretherapy height of +1.7 SD in comparison with +0.1 SD of PTPN11+ patients. Our data seem to confirm the hypothesis of a mild GH resistance in NS subjects caused by the SHP2 mutation.

Prader-Willi Syndrome

Clinical Signs The characteristic signs are short stature, hypotonia, hypogonadism, hyperphagia with progressive obesity, developmental delay (DD), behavior abnormalities, sleep disturbances and dysmorphic features. The clinical diagnosis is made according to scoring systems, recently revised. Other typical signs are short stature, respiratory problems and abnormalities in the autonomous regulation.

Etiology A deletion of a segment of the paternally derived chromosome 15q11-q13 is found in 70–75% of patients, 20–25% have a maternal disomy of the same region, 2–5% have imprinting defect mutations, and about 1% of patients a balanced translocation.

Growth Short stature is frequently observed in Prader-Willi syndrome (PWS). During the 1st year, growth is below the 3rd percentile, but later remains at the 10th percentile

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or below until 10 or 12 years of age. Thereafter, height velocity often declines due to a lack of growth spurt and a mean adult height of 155 cm for males and 148 cm for females have been reported. Specific growth charts for PWS have been prepared.

GH Secretion and Therapy In these patients, a dysregulation of the GH/IGF axis has been reported. In fact, a reduced spontaneous GH secretion to stimulation tests, and low serum IGF-1 and IGF-BP3 levels have been reported [11]. The reduced GH secretion together with the hypogonadotropic hypogonadism (retarded and incomplete sexual development), the hyperphagia and the high pain threshold suggest a hypothalamic-pituitary dysfunction for PWS patients. In 2000, the FDA approved the use of GH in these subjects. In several studies, the efficacy of GH treatment has been demonstrated. The initial positive effects on height velocity appear to be sustained throughout treatment. Moreover, many studies report that in patients treated for many years, growth continues to improve and TH-SDS can be reached. Long-term GH therapy shows a reduction in fat mass, an increase in muscle mass, and improved motor development. This was also documented in control studies in particular on younger patients [12]. The adverse effects reported for PWS patients are similar to those observed in GHD patients. The rapid growth associated with GH therapy may aggravate scoliosis. PWS is a condition with high risk factors for sudden death such as: upper airway obstruction and autonomic impairment of ventilatory control. It has been suggested that GH therapy may increase lymphoid and soft tissue inducing upper apnea events and may compromise pre-existing altered gas exchange with the augmented volume load. Furthermore, during GH therapy, hypopnea and central apnea events decreased in number and duration, as recorded by polysomnography. A recent review by Tauber et al. [13] showed that most deaths in PWS were due to respiratory infections and the causes of deaths were the same in treated and untreated PWS patients. Nevertheless, most cases of sudden death, in patients who received GH, occurred in the first months of treatment. Even though a causal relationship between GH therapy and sudden death has not been demonstrated, an accurate clinical protocol of management and follow-up of PWS patients at the start of therapy is mandatory.

Floating-Harbor Syndrome (Boston Floating Hospital and Harbor General Hospital [Pelletier et al., 1973])

Clinical Signs Floating-Harbor syndrome has as main features: short stature, developmental delay (DD), in particular speech, typical face: triangular with bulbous nose, prominent

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nasal bridge and broad columella, large nares and hypoplastic alae, deep-set eyes, wide mouth with thin lips. Normal occipitofrontal circumference (OFC) for height age.

Etiology Gene unknown.

Growth Small birth-length, during childhood very short stature (–4 to –6 SD), delayed bone age, normal puberty.

GH Secretion and Therapy Some cases of patients with GHD are reported [14, 15] with an increase in height velocity obtained with GH therapy. Nevertheless, there are no conclusive data as to the effect of GH therapy in this condition.

Personal Data We report a patient, 3.5 years of age, with severe short stature (-6.3 SD), typical face, bone age delay, GH deficit, who gained +2 SD with GH therapy over 5 years.

Kabuki Syndrome

Clinical Signs Minimal diagnostic criteria are DD, typical face: long palpebral fissures with eversion of the lateral part of the lower lids, epicanthal folds, short columella with depressed nasal tip, prominent or cupped ears, prominent filtrum, associated congenital anomalies: cleft palate, skeletal abnormalities, CHD (aortic coarctation, VSD, ASD, tetralogy of Fallot), cataract, optic nerve hypoplasia, hearing loss. Immune deficiency, autoimmune disorders.

Etiology Mostly sporadic. Various chromosomal abnormalities have been seen (interstitial duplication of 1p, del 6p, dup 12q).

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Growth At birth generally normal, postnatal short stature (< –2 SD) in 80% of cases.

GH Secretion and Therapy Some cases of GHD or neurosecretory dysfunction are reported [16]. Some cases have been treated with GH therapy with long-term good or moderate response [17]. The efficacy of GH therapy has not been proved in this syndrome. Isolated premature telarche and precocious puberty have been reported.

Personal Data Two patients (male ad female) showed GH deficiency and submitted to therapy with moderate results. The male suspended GH therapy due to autoimmune hemolytic anemia. The female showed early puberty treated with GnRH agonist.

Pallister-Hall Syndrome

Clinical Signs The clinical diagnostic criteria for PHS requires the presence of insertional polydactyly and hypothalamic hamartoma or isolated hamartoma or polydactyly in a relative of the proband. Other signs are bifid epiglottis, imperforate anus and other anomalies. This condition has variable clinical manifestations.

Etiology An autosomal dominant inheritance has been suggested. Isolated cases also occur. PHS is caused by mutations in the GLI3 gene located on chromosome 7 at 7p13. This gene codes a zinger finger domain DNA-binding protein expressed during embryogenesis. The mutations reported for PHS predict truncation of the protein C-terminal of the DNA-binding domain [18].

Growth Data of growth rate and final height are not available.

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GH Secretion and Therapy Different studies report a deficit in GH secretion in PHS patients [19, 20]. In particular, low mean and peak night-time GH and low levels of IGF-1 (neurosecretory dysfunction) have been reported. These findings suggest GH deficiency should be considered one of the causes of short stature in these patients, but the mechanism of GH deficiency associated with PHS is not clear. It has been hypothesized that the hypothalamic hamartoma determines hypothalamic dysfunction associated with low GH levels. Some adult patients presented normal stature and normal growth rate during childhood, so it suggests that in some cases GH decreases after childhood years or that other factors stimulate a normal growth.

Personal Data A female presented hypothalamic hamartoblastoma, postaxial polydactyly, genital anomalies, bifid epiglottis, neurosensorial deafness, and ASD. At 7.9 years she was 110 cm (<3rd centile), TH >50th centile; she showed GH deficiency at stimulation tests and low nocturnal GH concentration and still undergoes GH therapy. Her FH was 158.6 cm (25th centile for the GP). She presented normal growth spurt at puberty [18, 19] (fig. 3).

Silver-Russel Syndrome

Clinical Signs The main features are severe intrauterine and postnatal growth retardation, a typical facial appearance, limb asymmetry, fifth finger clinodactily and hemihypertrophy.

Etiology Most cases are sporadic (prevalence 1–30/100,000); genetic mutations have been identified as causes of Silver-Russel syndrome (SRS): chromosome 11p15 (epi)genetic mutations (35% of cases) and maternal uniparental disomy of chromosome 7 (10% of cases).

Growth SRS has been considered as a model of the role of imprinting in growth, human chromosome 11p15 contains a cluster of genes (IGF2 and CDKN1C) important for the

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Data 29/05/1998 26/10/1999 26/10/2000 26/10/2001

Altezza 157,00 158,10 158,10 158,10

Età 16,33 17,74 18,74 19,75

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Fig. 3. Growth curve and velocity of our patient with PHS treated with GH.

control of fetal and early childhood growth. Height SDS of children with UPD-7 decreased significantly during early childhood, and children with 11p15 epimutation are shorter and leaner than children with no detected genetic alteration. Mean birth length is –3 SDS, by the age of 4 years height SDS is of –3.5 to –4.4 and height is below and parallel to the 3rd centile and FH is –4.2 SDS (151.2 cm in males and 19.9 cm in females).

GH Secretion and Therapy Some studies reported that SRS patients have an impaired spontaneous secretion with reduced pulse frequency during the night [21]. Until now the contribution of GH/ IGF-1 axis defects on severe growth deficiency in SRS has not been determined and the genetic defects seem to play a more important role [22]. Very few data have been published on long-term therapy in SRS. There is an initial height gain comparable with small for gestational age (SGA).

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Personal Data In our study case, 7 of 9 SRS patients showed GH deficiency (4 partial and 3 severe) and were submitted to GH therapy. At the start of therapy, IGF-1 levels were below 1 SDS. Before puberty all the patients showed a good result in catch-up growth in particular an earlier beginning gave a better result. Nevertheless, the 3 subjects (2 males and 1 female) that reached the FH seldom arrived over the 3rd centile. The height gain obtained with GH was partially wasted at puberty for bone age advancement. The girls treated with GnRH agonist therapy for early puberty increased her height of 1 SD on pre-treatment height.

Skeletal Dysplasia

Skeletal dysplasias are genetic disorders that compromise linear growth and body proportion. It is a large group that includes more than 200 entities clinically distinct and genetically heterogeneous, in fact the genetic defect involves the collagen, matrix proteins, ion transporters and growth factor receptors. Most of the conditions are autosomal dominant; however, some are autosomal recessive. Their clinical diversity often makes these disorders difficult to diagnose and many attempts have been made to give a nosological classification to facilitate diagnosis. The most prevalent group is the achondroplasia group with achondroplasia (ACH) and hypochondroplasia (HCH) where the limbs are more affected than the trunk. We will consider also other skeletal dysplasias such as dyschondrosteosis (Leri-Weill syndrome), spondiloepiphyseal dysplasia (SED), pseudoachondroplasia (PSACH), Ellis-van-Creveld syndrome (EVC). The clinical evidence suggest that GH secretion or action is not responsible for growth deficiency in skeletal dysplasias, but some effects on growth acceleration have been reported. The deformities interfere with the efficacy of GH therapy. In skeletal dysplasias, differently than in GHD, height gain in the 1st year of therapy does not correlate with long-term efficacy. Moreover, GH given at an appropriated time can maintain height velocity within the normal range, even when the natural history of the condition suggests a decrease of height velocity. GH therapy associated to leg lengthening in some cases (ACH and HCH) may provide a better FH.

Personal Data In our series, 22 of 60 patients with skeletal dysplasia showed GH deficiency at pharmacological tests and were submitted to GH therapy: 6 ACH patients, 5 HCH, 7 SED, 3 PSACH, 1 Leri-Weill dyschondrosteosis and 1 EVC patient. Six patients reached the FH (2 ACH patients, 2 PSACH, 1 HCH and 1 EVC). In ACH subjects, H-SDS gain on baseline was

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+1.6SD on ACH growth charts, PSACH patients gained +1.4 SD on PSACH charts. In all the patients sitting height/height ratio showed no modification during therapy.

Achondroplasia

Clinical Signs Severe disproportional short stature with rizomelic shortening of legs and arms, macrocephaly with a prominent forehead, midface hypoplasia.

Etiology It is caused by mutations in the transmembrane region of the fibroblast growth factor receptor 3 (FGFR3). These mutations cause an increased FGFR3 activity that interferes with osteogenesis.

Growth Mean adult height in achondroplasia (ACH) is about 125 cm for women and 132 cm for men (–7SD below the average). Fetal growth is almost normal with birth length about –1.6 SD below the mean. Linear growth is fairly normal for the first postnatal months and later shows a profound decrease with a compromission of body proportions.

Growth Hormone Secretion Although clinical evidence suggests that patients with ACH have normal GH, some studies have reported a hypothalamic-pituitary dysfunction, including abnormal GH secretion with blunted response on different pharmacological tests and low IGF-1 levels.

Growth Hormone Therapy Given the skeletal abnormality, a reduced sensitivity to the action of GH and IGF-1 would be expected. Hence, supraphysiological GH doses have been used. There are several reports on GH therapy in ACH, although most involved few patients with a short-term follow-up. There are no definitive data available in these patients for FH and development in body proportions, in these patients. A number of short-term trials have reported that GH administration significantly increases the rate of growth.

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First year response is typically a 2- to 3-cm increase in growth velocity in prepubertal children (+0.5 SD height gain); GH treatment for up to 5 years can produce a total height gain of about +1 SD. In the subsequent years, a decrease in growth velocity was observed, but still greater than before therapy in most patients [23, 24]. This rapid desensitization to therapy may be due to abnormal functions of chondrocytes such as cell survival. Further studies are therefore essential to establish the duration of administration, dose and timing for the start of therapy. Sitting height SDS improved significantly during therapy, but body proportions did not show any significant change and this is a reassuring finding. In some studies, no bone age acceleration was observed under GH treatment; in contrast, other studies have reported a significant bone age acceleration. It has been found that OGTT was normal both before and after 12 months of GH treatment.

Hypochondroplasia

This is a genetic form of rhizomelic short stature with a wide variation in clinical expression.

Clinical Signs The patients do not have the typical macrocephaly seen in patients with ACH, and the incidence is difficult to determine since many mild forms remain undiagnosed. For the diagnosis of hypochondroplasia (HCH), the invariable radiological finding is the failure of increase in the vertebral interpedicular distance in the lumbar spine from L1 to L5.

Etiology The molecular defect is the same FGFR3 mutation seen in ACH and it was found in about 50% of HCH patients. The most prevalent mutation is the Asn540Lys. FGFR3 mutations activate the signaling of the receptor in the absence of ligand. The severity of the condition depends on the degree of the activation of the signaling pathway. Forms with a mild phenotype are often FGFR3 mutation negative.

Growth Mean adult height in HCH is 133–151 cm for women and 145–164 cm for men. The typical feature of many patients is the lack or the attenuation of growth spurt during puberty.

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Growth Hormone Secretion and Therapy Some authors reported a defect of IGF-1 in HCH patients. There are few studies on GH treatment in HCH patients. The height gain SDS was found to be of 1 DS after 2–3 years of treatment, but in some patients a gain of more than 3 SD has been reported. Some studies have shown that GH used earlier in childhood is not very effective, but others have shown better results if treatment is started at an early age. Moreover, it seems that the results are better when GH is administrated at puberty, when GH is able to regain the missing growth spurt [25]. In fact, the best responses were seen in patients treated when their puberty spurt failed. It is important to observe that pre-treatment height velocity in FGFR3 mutation-positive and mutation-negative patients is similar, but the response to GH treatment over 4 years is greater in the mutation-negative patients; moreover, in patients with the FGFR3 mutation a significant increase in sitting height has been reported which accentuated the disproportion. In many studies, no bone age acceleration was observed. GH treatment seems to be effective in patients with HCH even in severe forms more than in ACH patients. The difference is given by the difference in chondrocytes response to GH. In any case, FH data in these patients are necessary to confirm the effect of GH therapy.

Pseudoachondroplasia

Clinical Signs This is characterized by disproportionate very short stature, deformity of the legs, short fingers, ligamentous laxity, a waddling gait and early onset of osteoarthritis. Head size and facial features are normal.

Etiology A mutation in the cartilage oligomeric matrix protein (COMP) is found in 40% of the cases; the mutation interferes with the normal folding of the protein and causes an accumulation of the protein in the endoplasmic reticulum leading to the death of the cell and disrupts the formation of a normal extracellular matrix. This effect causes a decrease in cells in the bone matrix and a reduction in bone size. It is dominantly inherited.

Growth Final height is around 80–130 cm.

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Growth Hormone Secretion and Therapy Treatment of PSACH with GH has been reported in a small number of patients. In some cases, an empirical approach was used for individual patients. In this condition, the response to treatment was generally poor; in particular, in cases with deformities of the lower extremities GH therapy may worsen the deformities [25].

Ellis-van Creveld Syndrome

Clinical Signs ECV is a skeletal and ectodermal dysplasia characterized by short ribs, limbs, postaxial polydactyly, teeth and nails dysplasia and congenital heart defects (atrial septal defects). Etiology It is a rare autosomal-recessive disease with genetic heterogeneity. Mutations of the ECV1 and ECV2 genes (chr 4p16) have been identified as causative in this syndrome. Growth Short-limb dwarfism present at birth. Mean FH 109–152 cm. The association EVC and GH deficiency has been reported and GH therapy showed a satisfying effect on growth [26, 27]. Personal Data Our EVC patient with severe GH deficiency at pharmacological tests and spontaneous secretion showed a good growth gain with GH therapy during prepubertal age [27], but FH-SDS was just above the pretherapy height.

Dyschondrosteosis or Leri-Weill Syndrome

Clinical Signs Leri-Weill syndrome is a osteochondrodysplasia with mesomelic short stature and Madelung deformity of the wrist. Frequent signs are cubitus valgus, high-arched palate, and scoliosis.

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Etiology This skeletal disorder is caused by mutation or deletion of SHOX gene, a homeobox gene located at the pseudoautosomal region of the X and Y chromosome (SHOX haploinsufficiency or deficiency). Several mutations and deletions have been described. The phenotype of SHOX haploinsufficiency is highly variable ranging from short stature without dysmorphic signs to mesomelic skeletal dysplasia: Leri-Weill syndrome; the rare homozygous form referred to as Langer mesomelic dysplasia. SHOX regulates linear growth and may in part be involved in regulation of IGF-I gene expression or metabolism.

Growth The adult height in patients with Leri-Weill syndrome is variable but in most cases reduced, whereas the height reduction seems to be gender specific with a greater loss of height in females.

Growth Hormone Secretion and Therapy There are different studies on GH treatment in patients with Leri-Weill syndrome [28]. In these patients, an increase in height SDS and height velocity SDS over 24 months of therapy have been reported.

Discussion

Many developmental syndromes have short stature as a characteristic clinical feature. In recent years, the scientific community has shown an increased interest in these conditions. Advances in molecular genetics have contributed to the identification of the genetic etiology of a growing number of dysmorphic syndromes. The larger availability of recombinant GH has expanded the interest towards GH secretion and therapy to a wide variety of these conditions. In genetic syndromes growth defect is generally related to a deficit in cellular growth, caused by the underlying genetic defect and few of them are characterized by a severe disorder in GH secretion as a symptom of the disease. Nevertheless, in dysmorphic syndromes, some unknown abnormality in GH/ IGF-1 action may be hypothesized that contributes to the growth abnormality. For this reason, it is important to assess physiological growth hormone secretion, IGF-1 and IGFBP3 levels and therefore the detection of more subtle defects in GH secretion.

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Genetic syndromes, in particular when growth defect is prenatal, represent a natural model for further investigations in some unknown pathway of GH mechanism of action. On the other hand, also in physiological states the mechanisms that determine the achievement of an appropriate stature remain largely unknown. In dysmorphic syndromes with growth defect, the finding of a dysfunction in the GH/IGF-1 axis would permit a therapeutic attempt to obtain a higher FH than the expected height according to the natural history of the condition. The recent molecular studies on the etiology of Noonan syndrome have highlighted the link between the genetic defect of NS and the mechanisms of action of GH (JAK-STAT pathway). In NS, a partial GH insensitivity is due to the molecular defect found in more than 65% of the patients with this condition [9]. In the syndromes like PHS and PWS, where a defect in GH/IGF-1 axis is part of the syndrome, the benefit of GH-therapy is demonstrated on short-term-growth and also on FH [10]. In PWS the effect of GH extends to the general well-being: improvement of body composition, energy, muscle mass, cognitive functioning, decrease in hypotonia [12, 19, 20]. SRS represents a syndrome of IUGR associated with various dysmorphic features; in the mild forms the diagnosis may not be easy. In this condition, a defect in GH secretion, in particular in the nocturnal physiological profiles, are very common. GH therapy produces an increase in growth rate and does not worsen body asymmetry. Some authors suggest [21], although in a few patients, that this increase continues also when therapy had been stopped with an increase in FH as well. Differently from other authors, we found that height gain obtained with GH was partially wasted at puberty for bone age advancement, partially controlled by GnRH agonists therapy. Skeletal dysplasia is a large category of disorders that involve long bone and spine to a different extent, leading to disproportionate shortening. The molecular basis has been only recently identified in many of them and GH deficiency is not generally involved in the growth defect. Nevertheless, the experience of the beneficial effect of GH therapy in other conditions such as TS [5, 6, our data], without GH insufficiency, has led to clinical trials with supraphysiological doses. Some studies have reported an increase in growth velocity without conclusive data on FH [25]. This category of disorders includes so many conditions with different molecular defects, the number of treated patients is too little and FH data are indispensable for each group of skeletal dysplasia to obtain more conclusive results. Our experience of GH therapy in skeletal dysplasia is limited to the subjects that showed a GHD and could be treated according to the Italian healthcare laws. We found in ACH subjects who reached FH, a H-SDS gain on baseline of +1.6 SD on ACH growth charts and PSACH patients +1.4 SD on PSACH charts. In all the patients sitting height/height ratio showed no modification during therapy.

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Recognized Benefits of Growth Hormone Therapy

Therapeutic correction of partial GH deficiency and action in particular in some conditions such as PWS, SRS and NS. Improvement in growth velocity in prepuberty to reduce the difference with the age-matched children during schoolage. Gain of only 8 or 10 cm in height is of benefit to a child if it takes him into the bottom of the normal range and if his FH is moved from 120 to 130 cm. Benefit on the general well-being of the patients, e.g. in particular, modification of body composition, increase of energy, muscle mass, cognitive functioning, decrease in hypotonia.

Unsolved Problems

Systematic and accurate studies of the GH/IGF-1 axis are not available in most dysmorphic syndromes to detect subtle defects in GH secretion or a neuro-secretory dysfunction. The efficacious dosage of GH therapy has not been established for many conditions. Large databases, NCGS and KIGS, collected a high number of patients with dysmorphic syndromes, but FH data are available only for some less-rare conditions such as TS and PWS. Most of the trials are not randomized and in consideration of the small number of patients for each condition there are no defined homogeneous groups. Usual prediction methods are flawed in their application to children with syndromes: bone age estimation is often difficult and frequently pubertal growth spurt is blunted.

Complications and Warning

For some children with MR, GH therapy reduces the potential benefit even though there is an increase in FH. There is a cost for health care, for the family and child (daily injections, repeated visits to the hospital). In many rare diseases, the safety of GH therapy at supraphysiological doses has not been proven; in fact, cancer risk linked to the genetic defect and the natural history of many syndromes is not well known. Thus, care is needed with monitoring of IGF-1 and IGFBP-3.

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Conclusions

• Genetic syndromes may represent a natural model for further investigations in some unknown pathways of the GH mechanism of action. • A systematic and accurate study of the GH/IGF-1 axis may be important to detect subtle defects in GH secretion or a neurosecretory dysfunction. Some unknown abnormality in GH/IGF-1 action may be hypothesized contributing to the growth abnormality in dysmorphic syndromes. • Study of the GH/IGF-1 axis could help the definition of diagnostic criteria for more subtle forms of GHD that remain a dilemma also in nondysmorphic subjects. • To avoid bias in this difficult field, the use of specific growth charts to evaluate the effect of the therapy is advisable. • Long-term international multicenter studies with FH data will be mandatory to collect statistically significant groups of subjects for each syndromic condition. • In the future, the interest towards genetic syndromes and the skill in detecting very mild dysmorphic signs will permit a correct diagnosis in the large and sometimes not-well-defined group of idiopathic short stature.

References 1

2

3

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Mazzanti L, Nizzoli G, Tassinari D, Bergamaschi R, Magnani C, Chiumello G, Cacciari E: Spontaneous growth and pubertal development in Turner’s syndrome with different karyotypes. Acta Paediatr 1994;83:299–304. Pirazzoli P, Mazzanti L, Bergamaschi R, Perri A, Scarano E, Nanni S, Zucchini S, Gualandi S, Cicognani A, Cacciari E: Reduced spontaneous growth hormone secretion in patients with Turner’s syndrome. Acta Paediatr 1999;88:610–613. Wasniewska M, De Luca F, Bergamaschi R, Guarneri MP, Mazzanti L, Matarazzo P, Petri A, Crisafulli G, Salzano G, Lombardo F: Early treatment with GH alone in Turner syndrome: prepubertal catch-up growth and waning effect. Eur J Endocrinol 2004; 151:567–572. Davenport ML, Crowe BJ, Travers SH, Rubin K, Ross JL, Fechner PY, Gunther DF, Liu C, Geffner ME, Thrailkill K, Huseman C, Zagar AJ, Quigley CA: Growth hormone treatment of early growth failure in toddlers with Turner syndrome: a randomized, controlled, multicentric trial. J Clin Endocrinol Metab 2007;92:3406–3416.

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van Parenen YK, de Muinck Keizer-Schrama SMPF, Stijnen T, Sas TCJ, Jansen M, Otten BJ, HoorwedNijman JJG, Vulsma T, Stokvis-Brantsma WH, Rouwe CW, Reeser HM, Gerver WJ, Gosen JJ, Rongen-Westerlaken C, Drop SLS: Final height in girl with Turner syndrome after long-term growth hormone treatment in three dosages and low estrogens. J Clin Endocrinol Metab 2003;88:1119–1125. The Canadian Growth Hormone Advisory Committee: Impact of growth hormone supplementation on adult height in Turner syndrome: results of the Canadian Randomized Controlled Trial. J Clin Endocrinol Metab 2005;90:3360–3366. Osio D, Dahlgren J, Wikland KA, Westphal O: Improved final height with long-term growth hormone treatment in Noonan syndrome. Acta Paediatr 2005;94:1232–1237. Otten J, Noordam K: Short Stature in Noonan Syndrome: Results of Growth Hormone Treatments. Growth Hormone in Pediatrics – 20 years of KIGS. Basel, Karger, 2007, pp 347–355. Binder G, Neuer K, Ranke MB, Wittekindt NE: PTPN11 mutations are associated with mild growth Hormone resistance in individuals with Noonan Syndrome. J Clin Endocrinol Metab 2005;90:5377– 5381.

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10 Brown DC, Macfarlane CE, McKenna WJ, Patton MA, Dunger DB, Savage MO, Kelnar CJ: Growth hormone therapy in Noonan’s syndrome: non-cardiomyopathic congenital heart disease does not adversely affect growth improvement. J Pediatr Endocrinol Metab 2002;15:851–852. 11 Lee PDK, Allen DB, Angulo MA, Cappa M, Carrel AL, Castro-Magana M, Chiumello G: Consensus statement – Prader-Willi syndrome: growth hormone (GH)/Insulin-like growth factor axis deficiency and GH treatment. Endocrinologist 2000;10: 71S–73S. 12 Grugni G, Crinò A, Bosio L, et al: Genetic Obesity Study Group of Italian Society of Pediatric Endocrinology and Diabetology (ISPED). The Italian National Survey for Prader-Willi syndrome: an epidemiologic study. Am J Med Genet [A] 2008; 146:861–872. 13 Tauber M, Diene G, Molinas C, Hebert M: Review of 54 cases of death in children with Prader Willi syndrome. Am J Med Genet 2008;146A:881–887. 14 Wieczorek D, Wusthof A, Harms E, Meinecke P: Floating-harbor syndrome in two unrelated girls: mild short stature in one patient and effective growth hormone therapy in the other. Am J Med Genet 2001;104:47–52. 15 Cannavo S, Bartolone L, Lapa D, Venturino M, Almoto B, Violi A, Trimarchi F: Abnormalities of GH secretion in a young girl with Floating-Harbor syndrome. Ital Endocrinol Invest 2002;25:58–64. 16 Gabrielli O, Bruni S, Bruschi B, Carloni I, Coppa GV: Kabuki syndrome and growth hormone deficiency: description of a case treated by long-term hormone replacement. Clin Dysmorphol 2002;11: 71–72. 17 Tawa R, Kaino Y, Ito T, Goto Y, Kida K, Matsuda H: A case of Kabuki make-up syndrome with central diabetes insipidus and growth hormone neurosecretory dysfunction. Acta Paediatr Jpn 1994;36:412– 415. 18 Johnston JJ, Olivos-Glander I, Killoran C, et al: Molecular and clinical analyses of Greig cephalopolysyndactyly and Pallister-Hall syndromes: robust phenotype prediction from the type and position of GLI3 mutation. Am J Hum Genet 2005;76:609–622. 19 Zucchini S, Mazzanti L, Ambrosetto P, Salardi S, Cacciari E: Usual magnetic resonance imaging findings of the sella region in subjects with hypopituitarism: report of 4 cases. J Pediatr Endocrinol Metab 1998;11:35–44.

20 Galasso C, Scirè G, Fabbri F, Spadoni GL, Killoran CE, Biesecker LG, Boscherini B: Long-term treatment with growth hormone improves final height in a patient with Pallister-Hall syndrome. Am J Med Geent 2001;99:128–131. 21 Stanhope R, Albanese A, Azcona C: Growth hormone treatment of Russell-Silver syndrome. Horm Res 1998;49:37–40. 22 Binder G, Seidel AK, Martin D, Schweizer R, Schwarze C, Wollmann H, Heggerman T, Ranke M: The endocrine phenotype in Silver-Russell syndrome is defined by the underlying epigenetic alteration? J Clin Endocrinol Metab 2008;93:1402–1407. 23 Hertel NT, Eklöf O, Ivarsson S, Aronson S, Westphal O, Sipilä I, Kaitila I, Bland J, Veimo D, Müller J, Mohnike K, Neumeyer L, Ritzen M, Hagenäs L: Growth hormone treatment in 35 prepubertal children with achondroplasia: a five-year dose-response trial. Acta Paediatr 2005;94:1402–1410. 24 Tanaka H, Kubo T, Yamate T, Ono T, Kanzaki S, Seino Y: Effect of growth hormone therapy in children with achondroplasia: growth pattern, hypothalamic-pituitary function, and genotype. Eur J Endocrinol 1998;138:275–280. 25 Hertel T: Growth hormone treatment in skeletal dysplasia: the KIGS experience; in Ranke MB, Price DA, Reiter EO (eds): Growth Hormone Therapy in Pediatrics – 20 Years of KIGS. Basel, Karger, 2007, pp 356–368. 26 Pirazzoli P, Mazzanti L, Mandini M, Cau M, Ravagli L, Cacciari E: GH-deficiency in Ellis-vanCreveld Sindrome: Response to Replacement Therapy; in Bierich JR, Cacciari E, Raiti S (eds): Growth Abnormalities. Serono Symposia. New York, Raven Press, 1989, vol 56, pp 391–394. 27 Versteegh FG, Buma SA, Costin G, de Jong WC, Hennekam RC: Growth hormone analysis and treatment in Ellis-van Creveld syndrome, EvC Working Party. Am J Med Genet [A] 2007;143:2113–2121. 28 Blum WF, Crowe BJ, Quigley CA, Jung H, Cao D, Ross JL, Braun L, Rappold G: SHOX Study Group. Growth hormone is effective in treatment of short stature associated with short stature homeoboxcontaining gene deficiency: two-year results of a randomized, controlled, multicenter trial. J Clin Endocrinol Metab 2007;92:219–228.

Prof. Laura Mazzanti Department of Pediatrics, Rare Disease, Syndromology and Auxology Unit S. Orsola-Malpighi Hospital, University of Bologna Via Massarenti, 11, IT–40138 Bologna (Italy) Tel. +39 051 636 3723, Fax +39 051 636 3722, E-Mail [email protected]

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Growth Hormone-Resistant Syndromes: Long-Term Follow-Up Steven D. Chernausek Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, Okla., USA

Abstract Recombinant human IGF-I (rhIGF-I) has been approved as a therapeutic agent for short stature due to primary IGF-I deficiency following clinical trials that lasted more than 10 years. The first children treated with rhIGF-I were those with short stature resulting from defects in growth hormone (GH) action, either due to GH receptor abnormalities or the development of GH-neutralizing antibodies that arose following GH exposure. The administration of rhIGF-I to such children results in improvements in growth rate that are sustained over many years. This appears to improve adult height, but so far the effects are not as robust as when GH-deficient children are treated with human GH. Side effects are common but manageable and seldom necessitate discontinuation of therapy. Copyright © 2009 S. Karger AG, Basel

Children with growth hormone (GH) insensitivity were among the first to be treated with recombinant human IGF-I (rhIGF-I). The results of therapeutic trials in shortterm and long-term resulted in approval of rhIGF-I (Increlex®) for treatment of ‘primary IGF-I deficiency’ initially in the United States and later in Europe and other regions. Severe primary IGF-I deficiency is defined as a circulating concentration of IGF-I more than 3 SD below the mean for age and sex and height below –3 SD for age and sex in the face of normal nutrition and secretion of GH. It is also approved for treatment of short children with GH deficiency who develop high titers of GH- neutralizing antibodies following exposure to GH. IGF-I is established as an effective treatment for short stature due to GH insensitivity. It seemed logical to have some of the first trials in children be in those individuals with GH receptor deficiency or severe GH insensitivity. This was because there was no effective therapy for them and because, if the somatomedin hypothesis were correct, replacing IGF-I should effectively restore growth. However, it is useful to consider the questions faced by investigators at the onset of clinical studies. Would IGF-I, in fact, be anabolic and growth promoting over the long term? (If the direct

GH action was needed, rhIGF-I might be ineffectual in complete GH insensitivity.) What sort of dose and regimen would be best? Would the insulin-like effects of IGF-I prove problematic and limit its use? What unanticipated adverse effects would occur? Many of these questions have been answered by studies of several cohorts of children with GH insensitivity who received rhIGF-I therapy for various periods. Much of the data come from the largest and longest treated cohort of patients studied by the GH Insensitivity Syndrome (GHIS) Collaborative Group and it is that which is discussed here. Several other studies have been conducted with concordant results and these are cited as well.

Treatment Rationale

Growth hormone (GH) resistance is seen in its most robust form in complete GH receptor deficiency or Laron syndrome [1] These individuals are physiologically ‘blind’ to GH and suffer from extreme short stature, repetitive bouts of hypoglycemia, abnormalities of muscle and fat composition, and other metabolic disturbances. This occurs because production of IGF-I is substantially reduced and because direct actions of GH are not present. Individuals with GH insensitivity (defined as complete lack of GH effects as seen in homozygous null GH receptor states) are severely affected and potentially would benefit from therapies to overcome the GH resistance. Though GH insensitivity is rare, several studies show that there are intermediate forms of GH resistance. Thus, in a broad sense, methods to overcome GH resistance are needed.

Treatment Strategies

The most logical and time-tested therapeutic approach would be to restore normal concentrations of missing elements with appropriate distribution within the body. Individuals with GH insensitivity/resistance lack the direct actions of GH upon the skeleton and other tissues and the GH inducible factors IGF-I, IGF binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). For subjects in whom the GH receptor itself is missing or functionally abnormal, the most logical step would be to bypass the GH receptor, but activate the immediate downstream intracellular components (the jak-stat pathway). In theory this would be curative, but has the problem of potential lack of specificity since jak-stat pathways are used by other stimuli and, to date, there are no pharmaceuticals that might yield this effect. Because human GH (hGH) has been used for a long time and has shown a very good safety profile, supraphysiologic doses of hGH might be considered in certain forms of incomplete or partial GH resistance. In fact, the somewhat high doses of GH used to treat Turner syndrome, short children born SGA, and some cases of idiopathic short stature may reflect a degree of underlying GH resistance in these children. However, rhGH will not work in the

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severe forms of GH insensitivity and it is not necessarily true that extremely supraphysiologic doses of rhGH is the best approach in these individuals. Administration of IGF-I either alone or in combination with other circulating factors is also a logical approach to GH resistance because IGF-I is the principal mediator of the growth-promoting actions of GH and, theoretically, should promote growth in the absence of GH. IGF circulates in the blood as a ternary complex with IGFBP-3 and ALS. Since all are lowered in GH insensitivity, one might consider giving the entire complex back as the optimal therapy. Though there have been no attempts to use ternary complex treatment in humans, clinical trials have been conducted with an IGF-I /IGFBP-3 complex [2]. The potential advantages are a relatively prolonged half-life since ‘free’ IGF is more rapidly cleared from the circulation. Other advantages are the potential direct effects of IGFBP-3 and the possibility that residual ALS could complex with the IGFBP-3 and IGF-I, yielding the normal circulating form. However, because IGFBP-3 is a relatively large molecule, a substantial amount of protein needs to be given to deliver the quantity of IGF desired and the initial preparations required somewhat complex steps for the patient. Moreover, it is unclear whether the complex is absorbed intact (as complex) or whether IGF-I and IGFBP-3 separate and are absorbed at different rates following subcutaneous administration. Clinical trials using IGF-I/IGFBP-3 complex resulted in approval for use in severe primary IGF deficiency in the US, like rhIGF-I. However, litigious events resulted in its withdrawal from the US market. This leaves rhIGF-I as the only currently available therapy for GH resistance. The advantages of using rhIGF-I are that it is an active ligand for the type I IGF receptor (the receptor that mediates the growth promoting and metabolic actions of IGFI). Administration is simple with relatively rapid and complete absorption following subcutaneous injection. However, in the absence of IGFBP-3, the serum half-life of IGF-I is relatively short. Thus, twice daily injections are required in complete GH insensitivity.

Clinical Studies in Children with GH Insensitivity

Linear Growth Most patients with GH insensitivity have basal growth rates in the 2- to 3-cm/year range depending on their age. When rhIGF-I is given, growth promptly increases (see table) with the best growth occurring during the first year. In the large collaborative study when subjects were given at least 100 μg/kg b.i.d. for 2 years (n = 19) the growth velocity in the first year was 8.7 cm/year and in the second 6.1 cm/year [3]. This is equivalent to that reported in other studies [4–6]. Because the first-year growth rate is approximately 2 cm/year above the mean for age, there is some catch up in height. Thereafter, growth rates are typically normal for age, resulting in maintenance of height percentiles, but not much additional catch up growth.

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Table 1. Summary of published rhIGF-I clinical trials Trial

Ranke et al. [6] Klinger and Laron [5] Guevara-Aguirre et al. [4] Chernausek et al. [3]

Subjects

17 9 7 15 76

Height at start (SDS)

–6.6 –5.6 –8.0 –8.5 –6.5

rhIGF-I dose μg/kg

40–120 b.i.d. 175–200 daily 80 b.i.d. 120 b.i.d. 80–120 b.i.d.

Growth velocity cm/year baseline year 1

year 2

3.9 4.7 3.0 3.4 2.7

6.4 6.0 5.6 6.4 5.8

8.8 8.2 9.1 8.8 8.0

The effect of rhIGF-I therapy on final height is a natural question, but difficult to judge since no randomized controlled trial to final height has been conducted. Typical heights for adults with GH insensitivity syndrome are very low, and thus any meaningful increase, would be of benefit. One can estimate height gained in the small number of patients reported thus far by comparing their final stature with heights expected based on the growth charts developed by Laron et al. [7]. Final height data on 6 individuals were reported by the GHIS collaborative group, 3 of whom had received GnRH agonist treatment as well [3]. The duration of treatment was between 5.5 and 10 years. Using this relatively crude estimate, 5 of the 6 individuals gained more than 10 cm in height. The greatest apparent gain was seen in a male, who received therapy for nearly 8 years and had an estimated improvement in height of 23 cm without GnRH agonist treatment. Despite these encouraging results, most of these individuals did not achieve an adult height within the normal range. The reasons for this are multifactorial. First of all, nearly all the patients were far behind in height at the initiation of therapy. Indeed, all but one was below –4 SDs for height at the start. Secondly, patients with GH insensitivity syndrome, when given IGF appear not to grow as fast as severely GH deficient patients given GH [8]. The reasons for this are not completely clear, but may be attributed to relatively short half-life in the absence of IGFBP-3, restricted access of IGF-I to the growth plate, and the lack of GH direct effect.

Organ Growth Data from transgenic animals overexpressing IGF-I suggest that the kidney and spleen are particularly responsive to IGF-I. In patients with GH insensitivity, kidneys are typically small for age, even when accounting for height [3]. When rhIGF-I treatment is given, kidney length increases usually into the normal range. In a small number of patients, kidney size exceeds the norm but without any apparent adverse effect on

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250

Pretreatment On treatment

225 200

Glucose (mg/dL)

175 150 125 100 75 50 25 0 ⫺5 ⫺4 ⫺3 ⫺2 ⫺1

0

1

2

3

4

5

6

7

8

9

Day from rhIGF-1 initiation

Fig. 1. Blood glucose measures at treatment initiation, taken before lunch. Open circles represent before represent measures before treatment, and closed circles during rhIGF-I therapy. Dosing started at 40–60 μg/kg/dose b.i.d. and advanced to 120 μg/kg/dose. From Chernausek et al. [3], with permission.

kidney function. Similarly, spleen length is small for age, even when accounting for height, and usually normalizes in response to rhIGF-I.

Metabolic Effects There has been substantial concern that the hypoglycemia that occurs in patients with GH resistance might be aggravated with rhIGF-I treatment due to the insulinlike effects. Indeed, when rhIGF-I is given subcutaneously, circulating concentrations increase rapidly over 2 h and circulating blood glucose concentrations will usually fall unless carbohydrate is provided. Therefore, blood glucose concentrations were monitored frequently during the first clinical studies. This included in-hospital monitoring as well as home blood glucose measures. Figure 1 shows blood glucose measures from several patients before and during rhIGF-I initiation. Both hyper- and hypoglycemic episodes are evident prior to during rhIGF-I therapy. These results reflect the known underlying abnormalities of carbohydrate homeostasis seen in GH insensitivity [9,

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Table 2. Adverse events associated with rhIGF-I therapy Adverse event

Approximate frequency Management %

Comment

Hypoglycemia

50

administer rhIGF-I 30 min before meal

most hypoglycemia is due to underlying GH insensitivity; rhIGF-I induced hypoglycemia nearly always avoided when adequate carbohydrate intake follows injection

Lymphoid tissue growth resulting in hypoacusis, snoring, sleep apnea

20–25

tonsillectomyadenoidectomy; tympanostomy tube placement

may be more common with long-term exposure; hypoplasia of midface structures in GHIS may contribute

Hypertrophy at injection site

30

rotation of injection sites

Intracranial hypertension

4

temporarily discontinue rhIGF-I, restart at lower dose when ICH resolves

Coarsening of facial features

sporadic

improves with most often seen at end cessation of therapy at of therapy in final height conjunction with pubertal changes

also seen with hGH treatment.

Taken from Chernausek et al. [3].

10]. Though there have been clearly hypoglycemic events resulting from the administration of IGF-I, rhIGF-I injections, when given in conjunction with adequate meals, have little impact on glucose excursions. Plasma cholesterol is in the normal range for the majority and appeared to increase modestly over time. Similarly, there was a modest increase in triglycerides.

Effect on Body Composition Patients with GH insensitivity may have increased body fat compared to others and IGF-I may stimulate adipogenesis. Therefore, there has been some concern that rhIGFI may increase body fat. Studies by the collaborative group revealed modest changes in

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BMI over the course of treatment. Percent of body fat estimated by DEXA in 22 subjects showed body fat at 22% at baseline, a modest increase at 2 years, and then a return to the previous levels. Other studies have shown higher levels of body fat [11].

Adverse Events (table 2) The GHIS collaborative study found hypoglycemia to be the most common adverse event. However, this was usually related to the underlying GH insensitivity and not to the administration of rhIGF-I. Certain other adverse events appear due to growth of lymphoid tissue stimulated by IGF-I. These result from tonsillar and adenoidal hypertrophy, which led to hearing deficits in some and tympanostomy tube placement in 22% of the subjects. One third of the patients had lipohypertrophy at injection sites, but this was largely avoided with attention to site rotation. Intracranial hypertension, a known complication of rhGH therapy, also occurred with rhIGF-I therapy. The occurrence of this in three of the 76 subjects reported by the collaborative group suggests the incidence may be higher than with rhGH but such conclusions are limited by the small numbers. Another concern has been putative coarsening of facial features with rhIGF-I [12]. These changes were initially thought be similar to those seen in acromegaly, but subsequent studies involving measures of cephalometry indicate that this is not the case, but rather, result from soft tissue changes and increased soft tissue growth of the nose, lips, and other aspects of the face. This occurs somewhat sporadically and is difficult to quantify. There is improvement in this following withdrawal of rhIGF-I. Since patients with severe GH insensitivity have unusual facies, it is difficult to know to what to degree, if any, rhIGF-I altered there adult facial appearance.

Conclusion and Future Directions

Important areas for future research include efforts to optimization of therapy via dose changes, alternative regimens or different forms rhIGF-I, testing of rhIGF-I in less severe forms of GH resistance, and using GH and rhIGF-I in combination.

References 1

2

Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J: Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994;15:369–390. Camacho-Hubner C, Underwood LE, Yordam N, Yuksel B, Smith AV, Attie KM, Savage MO: Once daily rh IGF-1/rhIGFBP-3 treatment improves growth in children with severe primary IGF-I deficiency: results of a multicenter clinical trial. Proc Endocrine Soc 88th Ann Meet, Boston, 2006, abstr 40–1, p 132.

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Chernausek SD, Backeljauw PF, Frane J, Kuntze J, Underwood LE: Long-term treatment with recombinant insulin-like growth factor (IGF)-I in children with severe IGF-I deficiency due to growth hormone insensitivity. J Clin Endocrinol Metab 2007; 92:902–910.

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Guevara-Aguirre J, Rosenbloom AL, Vasconez O, Martinez V, Gargosky SE, Allen L, Rosenfeld RG: Two-year treatment of growth hormone (GH) receptor deficiency with recombinant insulin-like growth factor I in 22 children: comparison of two dosage levels and to GH-treated GH deficiency. J Clin Endocrinol Metab 1997;82:629–633. Klinger B, Laron Z: Three year IGF-I treatment of children with Laron syndrome. J Pediatr Endocrinol Metab 1995;8:149–158. Ranke MB, Savage MO, Chatelain PG, Preece MA, Rosenfeld RG, Wilton P: Long-term treatment of growth hormone insensitivity syndrome with IGFI: results of the european multicentre study. The working group on growth hormone insensitivity syndromes. Horm Res 1999;51:128–134. Laron Z, Lilos P, Klinger B: Growth curves for Laron syndrome. Arch Dis Child 1993;68:768–770. Blethen SL, Compton P, Lippe BM, Rosenfeld RG, August, GP, Johanson A: Factors predicting the response to growth hormone (GH) therapy in prepubertal children with gh deficiency. J Clin Endocrinol Metab 1993;76:574–579.

9 Brain CE, Hubbard M, Preece MA, Savage MO, Aynsley-Green A: Metabolic status of children with growth hormone insensitivity syndrome and responses to treatment with iIGF-I. Horm Res 1998;50:61–70. 10 Laron Z, Avitzur Y, Klinger B: Carbohydrate metabolism in primary growth hormone resistance (Laron syndrome) before and during insulin-like growth factor-I treatment. Metabolism 1995;44:113–118. 11 Laron Z, Ginsberg S, Lilos P, Arbiv M, Vaisman N: Body composition in untreated adult patients with Laron syndrome (primary GH insensitivity). Clin Endocrinol (Oxf) 2006;65:114–117. 12 Backeljauw PF, Underwood LE: Therapy for 6.5–7.5 years with recombinant insulin-like growth factor I in children with growth hormone insensitivity syndrome: a clinical research center study. J Clin Endocrinol Metab 2001;86:1504–1510.

Steven D. Chernausek Department of Pediatrics University of Oklahoma Health Sciences Center 1122 NE. 13th Street, Suite 1400, Oklahoma City, OK 73117 (USA) Tel. +1 405 271 2767, Fax +1 405 271 3439, E-Mail [email protected]

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Phenotypic Aspects of Growth Hormoneand IGF-I-Resistant Syndromes Martin O. Savagea ⭈ Alessia Davida ⭈ Cecilia Camacho-Hübnera ⭈ Louise A. Metherella ⭈ Adrian J.L. Clarka Department of Endocrinology, William Harvey Research Institute, Barts and the London, School of Medicine and Dentistry, Queen Mary’s, University of London, London, UK, and Karolinska Institute, Stockholm, Sweden

Abstract Major advances in the diagnosis and characterisation of growth hormone (GH) and IGF-I resistant disorders have occurred during the past 15 years. With these advances has come the realisation that there is broad phenotypic variation within these diagnostic categories. We discuss the current status of endocrine and molecular evaluation, focussing on the phenotypic characteristics of genetic Copyright © 2009 S. Karger AG, Basel defects in the GH-IGF-I axis.

Introduction

Growth hormone (GH) resistance states may exist in a range of clinical situations, both congenital and acquired. This chapter will be limited to disorders caused by genetic defects in the GH-IGF-I axis. GH-resistant syndromes have been recognised for over 40 years [1, 2]; however, the emergence of impaired IGF-I production and action is more recent [3, 4]. This chapter describes the different genetic defects in the GH-IGF-I axis and assesses their effects on phenotypic features and endocrine mechanisms. These disorders will be discussed principally in the paediatric context because the main presentation of affected children concerns reduced linear growth which may be pre-natal or post-natal or both.

Classification of Growth Hormone and IGF-I Resistance

The genetic disorders with resistance to the actions of GH or IGF-I are shown in table 1.

Table 1. Aetiological classification of GH insensitivity states Primary (genetic) defects of the GH-IGF-I axis 1 GH receptor defects a Extracellular mutations b Transmembrane mutations c Intracellular mutations 2 GH signal transduction defects (STAT 5b mutations) 3 IGF-binding protein defect 4 Acid-labile subunit defect 5 Bioinactive GH 6 Bioinactive IGF-I 7 Primary defects of IGF-I production or action a IGF-I gene mutations b IGF-I receptor mutations Secondary (acquired) dysfunction of the GH-IGF-I axis 1 Malnutrition, parenchymal liver disease, type 1 diabetes 2 Catabolic states (e.g. intensive care, post-operative) 3 Chronic inflammatory and nutritional disorders (e.g. juvenile chronic arthritis, Crohn’s disease) 4 GH neutralizing antibodies in patients with GH gene deletion

Classical Growth Hormone Resistance GH resistance resulting from a homozygous or compound heterozygous mutations in the GH receptor (GHR) is usually associated with the most severe clinical phenotype, known as Laron syndrome, after Zvi Laron, who first described the condition in 1966 [1, 2]. This disorder was also discovered in two rural communities in Ecuador, carrying the same GHR mutation [2]. Laron syndrome is seen principally in populations with a high rate of consanguinity and is prevalent in the Middle East [2] and the Indian sub-continent. A more heterogeneous series of patients, mainly resident in European countries was reported [5] and brought to light the heterogeneous nature of the paediatric GH-resistant phenotype. The phenotype of Laron syndrome resembles severe congenital GH deficiency, but with high levels of circulating GH [1]. The natural history of this disorder is characterised by very abnormal post-natal growth, resulting in extreme short stature in adult life with height ranging from –5 to –12 SD [3]. Fetal growth is relatively unaffected, although abnormal craniofacial development may be present at birth. Classical patients have a characteristic facies with mid-facial hypoplasia, which is not specific for Laron syndrome, also being seen in genetic GH deficiency and results from severe IGF-I deficiency. Other findings include poor musculature, delayed motor development, prominent forehead, laryngeal hypoplasia, hip dyplasia, osteopenia, thin skin, sparse and thin hair, and microphallus [1, 2]. In patients

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with Laron syndrome, relative obesity is present, with a decreased ratio of lean body mass to fat mass. Puberty is delayed, but normal reproductive function has been reported. The endocrine system, specifically the GH-IGF-I axis, is very disturbed. Serum IGF-I, IGFBP-3 and ALS are severely decreased and GH secretion is elevated, both basally and after stimulation. There are severe deficiencies in the GH-dependent proteins IGF-I, IGFBP-3, and acid-labile subunit (ALS) [2] serum GHBP is usually undetectable, associated with mutations in the extracellular domain of the GHR [5, 6]. However in 25% of cases, GHBP is normal [5] or even elevated [6].

Growth Hormone Receptor Mutations without Laron Syndrome The study of a large population of patients with primary GH resistance has demonstrated that some patients have a less severe phenotype [5]. In fact, there is a variation of phenotype, with heights ranging from –5 to –12 SD, even in the population of severely affected subjects from Ecuador who share the common E180 sp mutation [2]. It is clear however that some homozygous GHR mutations are associated with residual receptor function leading to generation of IGF-I and a milder phenotype. The phenotypic spectrum was demonstrated in a study which assessed facial appearance in subjects with unequivocal GH resistance. A range from typical Laron syndrome to normal facies was seen [7]. Within the same family, a wide variation of phenotype was seen between two siblings, who both had homozygous intracellular mutations in exon 10 of the GHR [8]. but whose adult heights were –8.3 and –5.6 SD, respectively.

Pseudo-Exon Growth Hormone Receptor Mutation A further GHR mutation which can be associated with a highly variable phenotype is an intronic pseudo-exon mutation, first reported by Metherell et al. [9] in 4 members of a consanguineous Pakistani family. This homozygous mutation causes the insertion of a pseudoexon between exons 6 and 7. The 108-bp insertion caused the addition, in-frame, of 36 amino acids between codons 206 and 207. We predicted that this would affect dimerization of the receptor; however, crystal structure modelling of this mutant GHR showed no alteration of the dimerization domain and cell studies resulting in a defect in trafficking [Ross, pers. commun.]. The 4 patients had unequivocal GH resistance with deficiencies of IGF-I, IGFBP-3 and ALS, but of a lesser degree than is usually seen in Laron syndrome. GH secretion was also elevated [10]. Height SD values ranged from –4.0 to –5.6. A larger number of patients have recently been published emphasizing the phenotypic variation [11].

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SH2 domain

DNA-binding domain 141

323

486

pY699

589

787

STAT 5b protein

A630P: Kofoed E 2003 1680delG: Hwa V 2007 N398fsX413: Hwa V 2005 Q368fsX376: Vidarsottir S 2006 R152X: Bernasconi A 2006

Fig. 1. Molecular defects of the STAT5b gene.

Dominant Negative Heterozygous Growth Hormone Receptor Mutation In 1997, Ayling et al. [12] provided a new insight into the genetics of GH resistance, describing the first heterozygous mutation with a dominant negative effect. The mutation (IVS8as-1 G→C) was situated in the acceptor splice site of intron 8 resulting in the skipping of exon 9 and the production of a truncated GHR. The mutant GHR formed heterodimers with the wild-type GHR and exerted a dominant negative effect on the normal protein. A second mutation (IVS9ds+1 G→A) leading to the same consequence was described in 2 Japanese siblings. We have also studied such a patient with a similar mutation which was present in the child’s mother and grandfather. The degree of short stature is milder than in classical GH resistance, height SDS ranging from –3.0 to –4.3, and facial features are usually normal. There is also a milder degree of IGF-I deficiency, with Ayling’s subject having a serum IGF-I of –2.4 SD [12]. All 4 subjects had normal GHBP.

STAT5b Mutations (fig. 1) Kofoed et al. [13] reported the first case of a homozygous mutation in exon 15 of the STAT5b gene and demonstrated that the mutant protein could not be activated by GH, therefore failing to activate gene transcription. This child had features of severe GH resistance together with immunodeficiency consistent with a non-functional STAT5b. Several more patients have now been reported. Most have immune deficiency. STAT5b appears to have an important role in gene expression induced

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by cytokines, such as interferon gamma Human defects in the STAT5b gene have recently been reviewed [14].

Idiopathic Short Stature Idiopathic short stature (ISS) refers to a heterogeneous group of short children with undefined aetiology. However, there is evidence of some degree of GH resistance in a sizeable proportion of children with significant short stature [15]. This has been elegantly demonstrated in studies of the IGF-I generation test in ISS patients and normal controls [16] A recent therapeutic trial demonstrated that it was necessary to treat ISS children with large doses of GH to achieve serum IGF-I levels of 2.0 SD [17]. However, true mutations of the GHR with evidence of decreased receptor function are rare [6].

Acid Labile Subunit Defect IGF-I, the key GH-dependent effector protein regulating human growth, circulates as a ternary complex consisting of IGF-I, IGFBP-3 and acid labile subunit (ALS). An ALS knock-out (KO) animal model provided new insights in the role of ALS in the IGF-I system, with growth deficits were seen 3 weeks after birth. In 2004, Domene et al. [18] reported the first human case of an inactivating ALS mutation. The defect was a guanine deletion at position 1338, resulting in a frame-shift and the appearance of a premature stop codon (1338delG, E35fsX120). The patient had minimal post-natal growth impairment but basal GH levels were increased associated with reduction in IGF-I and IGFBP-3 and undetectable ALS, unresponsive to stimulation by GH. A normal puberty growth spurt and final height has been documented in these patients, despite extreme deficiency of all GH-dependent peptides [19]. The characteristics of homozygous human ALS mutations have been further defined by recent reports, which now establish insulin resistance as an integral part of the phenotype.

IGF-I Gene Defects (fig. 2) The first human case of an IGF-I gene mutation was reported by Woods et al. [20] in 1996. The patient was a 15-year-old male who had severe intrauterine growth retardation (IUGR) and dysmorphic features consisting of microcephaly, micrognathia, ptosis, severe sensorineural deafness and intellectual retardation. Post-natal growth was also abnormal with a lack of response to GH therapy. The endocrine profile was of interest because serum IGF-I was undetectable, but IGFBP-3 was normal, thereby excluding a GHR defect. GH secretion was increased and ALS also slightly increased.

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Deletion of exons 4, 5

IGF-I gene

1

2

3

4

5

5’

6 3’

IGF-I V44M Bioinactive IGF-I

T/A exon 6 3’ UTR

IGF-I R36Q

Fig. 2. Molecular defects of the IGF-I gene.

The patient had insulin resistance with elevated fasting serum insulin. He was treated with recombinant human IGF-I for 2 years and showed a good growth response as well as normalising his insulin sensitivity. Molecular analysis showed a homozygous partial deletion of the IGF-I gene. Almost 10 years later, the second human case was reported by Wit’s group in Leiden, the Netherlands [21]. This patient also had intellectual retardation, deafness and a history of IUGR. Interestingly, his serum IGF-I was elevated indicating a biologically inactive IGF-I molecule. The molecular defect was a valine to methionine substitution at residue 44 of the mature IGF-I molecule. A third case has also been reported [3].

IGF-I Receptor Defects The key role of IGF-I in prenatal and post-natal growth has been confirmed by reports of mutations in the human IGF-I receptor (IGF-IR). This defect was first reported in 2 patients from Cincinnati and Leipzig [22]. Both had IUGR and post-natal growth failure. Four further cases, two sets of mother and daughter, have been described, all with some degree of IUGR and post-natal growth failure. It is likely that this defect is a rare cause of IUGR, although the degree of severity may depend on the degree of IGF-I signalling, which in turn is likely to be related to the nature of the molecular defect of the IGF-IR.

Conclusions

A number of general conclusions can be made concerning the phenotypes of patients with either GH or IGF-I resistance. Firstly, homozygous GHR mutations usually

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cause the phenotype of Laron syndrome, characterised by essentially normal prenatal growth but severely abnormal post-natal growth and final adult height. There is, however, a spectrum of phenotypical severity with some GHR defects causing a milder phenotype and less disturbance of the GH-IGF axis. Post-GHR defects such as mutations of the STAT5b gene also cause severe post-natal growth failure but with a subtly milder phenotype, not associated with the classical Laron syndrome facies. ALS mutations cause a failure of formation of the circulating ternary complex with extreme deficiency of circulating IGF-I, IGFBP-3 and ALS; however, the phenotype is mild and normal pubertal growth and final height have been reported. This may be explained by the relative normality, and even upregulation, of locally produced IGF-I, specifically in the growth plate. When the GHR is functional GH secretion may also be up-regulated resulting in insulin resistance. The key message concerning defects of the IGF-I gene and IGF-IR is that fetal growth is impaired, although to a variable degree. Post-natal growth is also abnormal. All these molecular defects are rare and the journey is just beginning of the characterisation of the range of phenotypes seen with each defect. The next decade will add more details and clarification of molecular mechanisms as more cases are diagnosed and genetic investigation becomes more refined.

References 1

2

3

4

5

6

Laron Z: Laron syndrome (primary growth hormone resistance or insensitivity): the personal experience 1958–2003. J Clin Endocrinol Metab 2004;89: 1031–44. Rosenfeld RG, Rosenbloom AL, Guevara-Aguirre J: Growth hormone (GH) insensitivity due to primary GH receptor deficiency. Endocr Rev 1994;15:369– 390. Woods KA: Genetic defects of the growth hormoneIGF axis associated with GH insensitivity. Endoc Dev 2007;11:6–15. Walenkamp MJ, Wit JM. Genetic disorders in the GH IGF-I axis in mouse and man. Eur J Endocrinol 2007;157(suppl 1):S15–26. Woods KA, Dastot F, Preece MA, Clark AJ, PostelVinay MC, Chatelain PG. Ranke MB, Rosenfeld RG. Amselem S, Savage MO: Phenotype: genotype relationships in growth hormone insensitivity syndrome. J Clin Endocrinol Metab 1997;82:3529– 3535. Savage MO, Attie KM, Camacho-Hübner C, David A, Metherell LA, Clark AJL: Investigation and treatment of patients with characteristics of growth hormone insensitivity. Nat Clin Pract Endocrinol Metab 2006;2:395–407.

7 Burren CP, Woods KA, Rose SJ, Tauber M, Price DA, Heinrich U, Gilli G, Razzaghy-Azar M, Al-Ashwal A, Crock PA, Rochiccioli P, Yordam N, Ranke MB, Chatelain PG, Preece MA, Rosenfeld RG, Savage MO: Clinical and endocrine characteristics in atypical and classical growth hormone insensitivity syndrome. Horm Res 2001;55:125–130. 8 Milward A, Metherell L, Maamra M, Barahona MJ, Wilkinson IR, Camacho-Hubner C, Savage MO, Strasberger CJ, Clark AJL, Ross RJM, Webb SM: Growth hormone (GH) insensitivity syndrome due to a GH receptor truncated after Box1, resulting in isolated failure of STAT 5 signal transduction. J Clin Endocrinol Metab 2004;89:1259–1266. 9 Metherell LA, Akker SA, Munroe PB, Rose SJ, Caulfield M, Savage MO, Chew SL, Clark AJL: Pseudoexon activation as a novel mechanism for disease resulting in atypical growth hormone insensitivity. Am J Hum Genet 2001 69:641–646. 10 Bjarnason R, Banerjee K, Rose SJ, Rosberg S, Metherell L, Clark AJL, Albertsson-Wikland K, Savage MO: Spontaneous growth hormone secretory characteristics in children with partial growth hormone insensitivity. Clin Endocrinol 2002;57: 357–361.

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11 David A, Camacho-Hubner C, Bhangoo A, Rose SJ, Miraki-Moud F, Akker SA, Butler GE, Ten S, Clayton PE, Clark AJL, Savage MO, Metherell LA: An intronic growth hormone receptor mutation causing activation of a pseudoexon is associated with a broad spectrum of growth hormone insensitivity phenotypes. J Clin Endocrinol Metab 2007; 92;655–659. 12 Ayling RM, Ross R, Towner P, Von Laue S, Finidori J, Moutoussamy S, Buchanan CR, Clayton PE, Norman MR: A dominant-negative mutation of the growth hormone receptor causes familial short stature. Nat Genet 1997;16:13–14. 13 Kofoed EM, Hwa V, Little B, Woods KA, Buckway CK, Tsubaki J, Pratt KL, Besrodnik L, Jasper H, Tepper A, Heinrich JJ, Rosenfeld RG: Growth hormone insensitivity associated with a STAT5b mutation. N Engl J Med 2003;349:1139–1147. 14 Rosenfeld RG, Belgorosky A, Camacho-Hübner C, Savage MO, Wit JM: Defects in growth hormone receptor signalling. Trends Endocrinol Metab 2007; 18134–18141. 15 Wit JM, Reiter EO, Ross JL, Saenger PH, Savage MO, Rogol AD, Cohen P: Idiopathic short stature: management and growth hormone treatment. Growth Horm IGF Res 2008;18;111–136. 16 Selva KA, Buckway CK, Sexton G, Pratt KL, Tjoeng E, Guevara-Aguirre J, Rosenfeld RG: Reproducibility in patterns of IGF generation with special reference to idiopathic short stature. Horm Res 2003;60:237– 246. 17 Cohen P, Rogol AD, Howard CP, Bright GM, Kappelgaard AM, Rosenfeld RG, American Norditropin Study Group: Insulin growth factorbased dosing of growth hormone therapy in children: a randomized, controlled study. J Clin Endocrinol Metab 2007;92:2480–2486.

18 Domene H, Bengolea SV, Martinez AS, Ropelato MG, Pennisi P, Scaglia P, Heinrich JJ, Jasper HG: Deficiency of the circulating insulin-like growth factor system associated with inactivation of the acid-labile subunit gene. N Engl J Med 2004;350: 570–577. 19 Domené HM, Martínez AS, Frystyk J, Bengolea SV, Ropelato MG, Scaglia PA, Chen JW, Heuck C, Wolthers OD, Heinrich JJ, Jasper HG: Normal growth spurt and final height despite low levels of all forms of circulating insulin-like growth factor-I in a patient with acid-labile subunit deficiency. Horm Res 2007;67:243–249. 20 Woods KA, Camacho-Hübner C, Savage MO, Clark AJL: Intrauterine growth retardation and post-natal growth failure associated with deletion of the insulin-like growth factor-I gene. N Engl J Med 1996;355:1363–1367. 21 Walenkamp MJ, Karperien M, Pereira AM, HilhorstHofstee Y, van Doorn J, Chen JW, Mohan S, Denley A, Forbes B, van Duyvenvoorde HA, van Thiel SW, Sluimers CA, Bax JJ, de Laat JA, Breuning MB, Romijn JA, Wit JM: Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. J Clin Endocrinol Metab 2005;90:2855– 2864. 22 Abuzzahab MJ, Schneider A, Goddard A, Grigorescu F, Lautier C, Keller E, Kiess W, Klammt J, Kratzsch J, Osgood D, Pfäffle R, Raile K, Seidel B, Smith RJ, Chernausek SD, Intrauterine Growth Retardation (IUGR) Study Group: IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med 2003;349:2211–2222.

Prof. Martin Savage Department of Endocrinology John Vane Science Centre, Charterhouse Square London EC1M 6BQ (UK) Tel. +44 20 7882 6233, Fax +44 20 7882 6234, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 151–166

Double Diabetes: A Mixture of Type 1 and Type 2 Diabetes in Youth Paolo Pozzilli ⭈ Chiara Guglielmi Department of Endocrinology and Diabetes, University Campus Bio-Medico, Rome, Italy

Abstract The increase in the incidence of type 1 diabetes (T1D), especially in children <5 years of age reported over the past decade can be attributed to changes in environmental factors, either quantitative or qualitative, rather than to an effect of genetic factors operating in such a short period of time. The notable increase in the incidence of type 2 diabetes (T2D) in children and adolescents is very likely the consequence of the increase in obesity and sedentary life style occurring in developed countries. The increase in the number of children and adolescents with a mixture of the two types of diabetes has recently come to light (i.e. subjects who are obese and/or with signs of insulin resistance as well as positive for markers of autoimmunity to β cells). Under the current classification, it is difficult to define the type of diabetes affecting these young subjects, being classified as T2D because they are obese and insulin resistant, but also as T1D because of the presence of auto-antibodies to β cells. There is no doubt that these subjects show an overlapping diabetes phenotype typical of both T1D and T2D suggesting that the current classification of diabetes should be revised taking into account this new form of diabetes which has called double diabetes or hybrid diabetes. Copyright © 2009 S. Karger AG, Basel

In the latest classification of diabetes [1] the terms type 1 diabetes (T1D) and type 2 diabetes (T2D) were introduced to replace insulin-dependent and non-insulindependent diabetes, respectively, thus reflecting with the new nomenclature two distinct forms of the disease in terms of pathogenesis. Since then a number of studies have suggested that differences between the two forms of diabetes are not always straightforward and in many cases common pathogenic processes may operate [2]. Not surprisingly, this has meant questioning the present classification of diabetes and the provocative proposal of declassifying this disease [3]. Even the best animal model of T1D (i.e. the NOD mouse) has some genetic background that can predispose these mice to insulin resistance before the destruction of β cells and in absence of hyperglycemia [4]. These observations suggest that nonimmunological processes may also

be important in the cascade of events leading to β cell destruction and, conversely, an immune-mediated process can accelerate β cell failure in T2D. Whatever the arguments, both forms of diabetes are on the increase in nearly all countries; T1D is the most prevalent chronic disease in childhood and T2D is now reaching the proportion of an epidemic worldwide. The increase in incidence of T1D in the past decade, especially in children under the age of 5 years [5], can be attributed to changes in environmental factors either quantitative or qualitative, but is unlikely to be an effect of genetic factors in such a short period. The increase in the incidence of T2D in children and adolescents is more likely to be caused by the increase in obesity and sedentary lifestyles in developed countries [6, 7]. Youths with T2D show features of insulin resistance (obesity, acanthosis nigricans, high insulin/C-peptide levels, polycystic ovarian syndrome in girls) and typically a family history of T2D [8]. From a clinical standpoint their hyperglycemia is mild, ketosis is rare, and the management of hyperglycemia includes diet and oral hypoglycemic agents. Moreover, the increase in the number of children and adolescents with a mixture of the two types of diabetes has been recently brought to our attention, i.e. subjects who are obese and/or with signs of insulin resistance as well as being positive for markers of autoimmunity to β cells.

Diabetes and Obesity

Diabetes and obesity are twin interrelated epidemics which threaten to engulf the world’s healthcare systems over the next two decades. The prevalence of both is increasing at an alarming rate with up to 400 million people likely to develop diabetes in the next 15–20 years unless action is taken. The figures for obesity are even more disturbing with prevalence in children showing huge increases. Whereas diabetes was a rare disease in the developing world 50 years ago, rates are now soaring even in the poorest countries. Much of this increase in diabetes prevalence is directly attributable to the epidemic of obesity. If no one were overweight or obese, the prevalence of T2D would be around 1% compared with the actual rate of 4% in the UK and more than 10% in many countries today [9]. Both diabetes and obesity are associated with significant mortality and morbidity from macrovascular disease while diabetes carries the extra burden from the specific microvascular complications of retinopathy, neuropathy and nephropathy. Diabetes and obesity are on the rise at a rapid rate. Back in 1994, it was predicted that there would be 239 million people with diabetes by 2010. Today, 2 years before that date, the figure has already been significantly outstripped, with some 246 million people worldwide living with diabetes [10]. Much of this is driven by the obesity epidemic. The World Health Organization (WHO) estimates that there are currently 1.6 billion adults (aged >15 years) who are overweight and at least 400 million adults who are clinically obese. The outlook for

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the future is even bleaker, with more than 700 million people predicted to be obese by 2015 and a total of 380 million people predicted to be living with diabetes by the year 2025 [11, 12]. Each year a further seven million people develop diabetes; that is two people every 10 s [13].

Obesity in Childhood, Adolescence and Adulthood

Since the mid-1970s, the prevalence of overweight and obesity has increased sharply for both adults and children. Data from two NHANES surveys show that among adults aged 20–74 years the prevalence of obesity increased from 15.0% (in the 1976–1980 survey) to 32.9% (in the 2003–2004 survey). The two surveys also show increases in overweight among children and teens. For children aged 2–5 years, the prevalence of overweight increased from 5.0 to 13.9%; for those aged 6–11 years, the prevalence increased from 6.5 to 18.8%, and for those aged 12–19 years, the prevalence increased from 5.0 to 17.4%. These increasing rates raise concern because of their implications for Americans’ health [14]. Although one of the national health objectives for the year 2010 is to reduce the prevalence of obesity among adults to less than 15%, current data indicate that the situation is worsening rather than improving.

Definitions for Adults For adults, overweight and obesity ranges are determined by using weight and height to calculate a number called the ‘body mass index’ (BMI). BMI is used because, for most people, it correlates with their amount of body fat. An adult who has a BMI between 25 and 29.9 is considered overweight. An adult who has a BMI of 30 or higher is considered obese. It is important to remember that although BMI correlates with the amount of body fat, BMI does not directly measure body fat. Other methods of estimating body fat and body fat distribution include measurements of skinfold thickness and waist circumference, calculation of waist-to-hip circumference ratios, and techniques such as ultrasound, computed tomography, and magnetic resonance imaging (MRI).

Definition for Children A child’s weight status is determined based on an age- and sex-specific percentile for BMI rather than by the BMI categories used for adults. Classifications of overweight for children and adolescents are age- and sex-specific because children’s body

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Table 1. Prevalence of overweight among US children and adolescents (age 2–19 years) Age

2–5 years 6–11 years 12–19 years

Survey periods NHANES I 1971–1974

NHANES II 1976–1980

NHANES III 1988–1994

NHANES 2003–2004

5 4 6.1

5 6.5 5

7.2 11.3 10.5

13.9 18.8 17.4

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

composition varies as they age and varies between boys and girls. The Center for Disease Control and Prevention defined overweight as at or above the 95th percentile of BMI for age and ‘at risk for overweight’ as between the 85th and 95th percentile of BMI for age [15, 16]. European researchers classified overweight as at or above the 85th percentile and obesity as at or above the 95th percentile of BMI [17]. Overweight children and adolescents are at risk for health problems during their youth and as adults. For example, during their youth, overweight children and adolescents are more likely to have risk factors associated with cardiovascular disease (such as high blood pressure, high cholesterol, and T2D) than are other children and adolescents [18]. Overweight children and adolescents are more likely to become obese as adults [19, 20]. For example, one study found that approximately 80% of children who were overweight at age 10–15 years were obese adults at age 25 years. Another study found that 25% of obese adults were overweight as children [21]. The latter study also found that if overweight begins before 8 years of age, obesity in adulthood is likely to be more severe.

Trends in Childhood Overweight

In the middle of the 1990s the average prevalence of overweight, including obesity, for youth in European countries was about 22%. The highest level was in Italy, with more than 30% affected children [22]. The highest prevalence of obesity among schoolage children (10–16 years of age) was in Malta (7.9%) and the lowest in Lithuania and Latvia (0.4%) [23]. Tables 1–3 and figures 1–3 show trends in childhood overweight based on NHANES data for various age groups, beginning with NHANES I (1971–1974) and ending with NHANES 2003–2004. Data from NHANES I (1971–1974) to NHANES 2003–2004 show increases in overweight among all age groups [24, 26]:

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Table 2. Adolescent boys prevalence of overweight by race/ethnicity (age 12–19 years) Survey periods

Non-Hispanic white Non-Hispanic black Mexican-American

NHANES III 1988–1994

NHANES 2003–2004

11.6 10.7 14.1

19.1 18.5 18.3

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

Table 3. Adolescent girls prevalence of overweight by race/ethnicity (age 12–19 years) Survey periods

Non-Hispanic white Non-Hispanic black Mexican-American

NHANES III 1988–1994

NHANES 2003–2004

7.4 13.2 9.2

15.4 25.4 14.1

Sex-and age-specific BMI ≥95th percentile based on the CDC growth charts. All values given in percent [24–26].

• among preschool-age children (2–5 years), the prevalence of overweight increased from 5.0 to 13.9% • among school-age children (6–11 years), the prevalence of overweight increased from 4.0 to 18.8% • among school-age adolescents (12–19 years), the prevalence of overweight increased from 6.1 to 17.4%. Although overweight has increased for all children and adolescents over time, NHANES data indicate disparities among racial/ethnic groups. Figures 1–3 compare the prevalence for racial/ethnic groups of adolescent boys and girls aged 12 through 19 years. The prevalence rate of overweight was slightly higher among adolescent non-Hispanic white boys (19.1%) than among non-Hispanic black boys (18.5%) and MexicanAmerican boys (18.3%) [25]. Data from NHANES III (1988–1994) through NHANES 2003–2004 showed that adolescent non-Hispanic white and black boys experienced larger increases in the

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20 Percent

15 10 5

18.8

Age 2–5 years Age 6–11 years Age 12–19 years 5.0

4.0

6.1

17.4

13.9 11.3 10.5 5.0

6.5

7.2 5.0

0 NHANES I 1971–1974

NHANES II 1976–1980

NHANES III 1988–1994

NHANES 2003–2004

Survey period

Fig. 1. Prevalence of overweight among US children and adolescents (age 2–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI >95th percentile based on the CDC growth charts [24–26].

prevalence of overweight (7.5% and 7.8%, respectively) compared to the increase among Mexican American boys (4.2%): • among non-Hispanic white boys, the prevalence of overweight increased from 11.6 to 19.1% • among non-Hispanic black boys, the prevalence of overweight increased from 10.7 to 18.5% • among Mexican-American boys, the prevalence of overweight increased from 14.1 to 18.3%. Non-Hispanic black girls had the highest prevalence of overweight (25.4%) compared to that of non-Hispanic white (15.4%) and Mexican-American (14.1%) girls [25]. Data from NHANES III (1988–1994) through NHANES 2003–2004 showed that non-Hispanic black adolescent girls experienced the largest increase in the prevalence of overweight (12.2%) compared to non-Hispanic white adolescent (8.0%) and Mexican-American adolescent (4.9%) girls [24, 26]: • among non-Hispanic white girls, the prevalence of overweight increased from 7.4 to 15.4% • among non-Hispanic black girls, the prevalence of overweight increased from 13.2 to 25.4% • among Mexican American girls, the prevalence of overweight increased from 9.2 to 14.1%.

Causes of Overweight and Obesity

Obesity is a complex condition with genetic, metabolic, behavioral and environmental factors all contributing to its development. However, the dramatic increase in the

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Non-Hispanic white

Fig. 2. Adolescent boys prevalence of overweight by race/ ethnicity (age 12–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI >95th percentile based on the CDC growth charts [24–26].

Percent

30

Non-Hispanic black Mexican-American 19.1

20 11.6

10.7

14.1

18.5

18.3

10 0 NHANES III 1988–1994

NHANES 2003–2004 Survey period

Non-Hispanic white

Fig. 3. Adolescent girls prevalence of overweight by race/ ethnicity (age 12–19 years). National Health and Nutrition Examination Surveys. Sex-and age-specific BMI > 95th percentile based on the CDC growth charts [24–26]..

Percent

30

Non-Hispanic black Mexican-American

20 10

15.4

13.2 7.4

25.4 14.1

9.2

0 NHANES III 1988–1994

NHANES 2003–2004 Survey period

prevalence of obesity in the past few decades can only be attributed to significant changes in lifestyle influencing both children and adolescents [12]. These obesitypromoting environmental factors are usually referred to today under the general term of ‘obesogenic’ or ‘obesigenic’ [13]. The current changing nature of this ‘obesogenic’ environment has been well described in a WHO Technical Report as follows: ‘Changes in the world food economy have contributed to shifting dietary patterns, for example, increased consumption of energy-dense diets high in fat, particularly saturated fat, and low in unrefined carbohydrates. These patterns are combined with a decline in energy expenditure that is associated with a sedentary lifestyle, motorized transport, labor-saving devices at home, the phasing out of physically demanding manual tasks in the work-place, and leisure time that is preponderantly devoted to physically undemanding pastimes.’ Under the notion ‘lifestyle’ are included dietary changes and cultural, environmental, social and economic factors. Therefore, apart from genetic factors, the prerequisite for becoming obese is an imbalance between energy expenditure, modulated primarily by physical inactivity, and energy intake from excessive food and drinks.

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Consequences of Overweight and Obesity

Childhood overweight is associated with various health-related consequences. Overweight children and adolescents may experience immediate health consequences and may be at risk for weight-related health problems in adulthood. Being overweight or obese increases the risk of many diseases and health conditions, including the following: hypertension, osteoarthritis, dyslipidemia, T2D, coronary heart disease, stroke, gallbladder disease, sleep apnea and some cancers (endometrial, breast, and colon).

Double Diabetes

The attractive term ‘double diabetes’ (DD) applied to the pediatric diabetic population was first introduced by Libman and Becker and coworkers [27, 28] when referring to subjects with an atypical form of diabetes, also called hybrid diabetes, type 1.5 diabetes or LADY (latent autoimmune diabetes in youth). The issue of DD was recently revised by two investigators of the present application [29]. The presence of autoimmune markers towards β cells, namely glutamic acid decarboxylase (GAD), thyrosine phosphatase antibodies (IA-2) and insulin autoantibodies (IAA), typically define cases of DD in patients with T2D [30, 31]. It is difficult to define what type of diabetes these subjects suffer from under the current classification, being classified as affected by T2D because they are obese and insulin resistant but also as affected by T1D because of presence of autoantibodies to β cells. There is no doubt that these subjects present with an overlapping phenotype of both T2D and T1D. In the adult population, these subjects are usually defined as affected by latent autoimmune diabetes in the adult (LADA) [32–34]. Such definition is generally based on autoantibody positivity, age at onset (>35 years) and insulin-independence following diagnosis of hyperglycemia for a period of at least 6 months. Several studies have demonstrated a more aggressive course of the disease in LADA subjects characterized by failure of oral hypoglycemic therapy and progressive β cell loss leading to insulin dependency usually within 5 years of diagnosis in subjects with more than one autoantibody to β cells and when aged 35–45 years [35]. LADA is found in approximately 10% of all cases of T2D; however, insulin resistance and obesity are not main features of LADA subjects [36] whereas they are in DD. Therefore, it looks like LADA represents one end of a rainbow of autoimmune diabetes which is distinguishable from classical T1D only because it is diagnosed in adulthood and presents with some clinical, anthropometric and metabolic features usually associated with T2D [35]. Despite obesity and metabolic syndrome being on the increase, in particular among Hispanic and African-Americans but also in Caucasian youths [37], we know very little about the prevalence of DD. The prevalence and significance of autoimmune markers in children who clinically present with T2D needs to be established in different populations.

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In particular, it needs to be established whether autoantibody-positive youths with DD progress more rapidly to insulin dependence than those with T2D without autoantibodies to β cells. This is also relevant because these youths may be at increased risk for complications associated with loss of β cell function, including hypoglycemia, ketoacidosis, difficult management of the disease and microvascular complications [38], in addition to macrovascular complications typically associated with T2D.

Role of Genes in Double Diabetes

It is accepted that both genetics and the environment play a role in T1D as demonstrated by the observation that disease concordance among identical twins is 30–50% [39, 40]. It has been calculated that the HLA region accounts for approximately 40% of the genetic risk, although other genes have an additional risk [41]. Amongst the putative environmental factors for T1D viral infections, infant dietary exposure, deficiency in vitamin D supplementation, and reduced number of infections accounting for the hygiene hypothesis, have been proposed [42–44]. In T2D, the role of genetic causative factors, although they have been only recently elucidated in determining the disease, is probably limited without the ‘weight’ of the environmental factors which include overnutrition, sedentary lifestyle and obesity [45]. The role of genes in DD is certainly of interest. One may argue that the major genes independently associated with susceptibility to either T1D, e.g. HLA, insulin gene VNTR, protein tyrosine phosphatase non-receptor (PTPN22), cytotoxic T -lymphocyte-associated antigen-4, (CTLA-4) [reviewed in 46], or T2D, e.g. adiponectin gene (APM1), plasma cell membrane glycoprotein 1 (PC-1) gene, transcription factor 7-like 2 (TCF7L2) gene [reviewed in 47] can represent genetic determinants for DD. We can speculate that in DD the frequency of the major T1D genetic susceptibility gene (HLA) may be reduced whereas an association exists with genes associated with T2D risk. Most interesting are genes that in view of their function may influence the pathogenic processes operating both in T1D and T2D potentially leading to DD.

Role of Environmental Factors in Double Diabetes

The environmental factors which influence both T1D and T2D disease processes may indeed play an important role in DD. A worldwide obesity epidemic as a consequence of current lifestyle influences the T2D process, but how such a condition or other factors associated with obesity affect or modulate the process leading to β cell destruction is unknown. Surprisingly, an increase in body mass index (BMI) has been reported in one third of children at diagnosis of diabetes [48], a form of clinical presentation which was never reported in the past for diabetes diagnosed in childhood. Several hypotheses have been put forward, the most widely accepted being linked to

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the increased risk for developing T1D in subjects who are overweight during childhood or experienced rapid early growth. The ‘accelerator hypothesis’ argues that T1D and T2D are the same disorder of insulin resistance set against different genetic background and predicts a general inverse relationship between BMI (surrogate for insulin resistance) and age at diagnosis of T1D [49]. The accelerator hypothesis identifies three accelerators that determine the rate at which β cell mass fails during life represented by constitution (genes/gestation), insulin resistance (lipotoxicity/antigenicity) and immune response genotype (HLA) [49]. There is still controversy about the accelerator hypothesis, the major concern being at what point obesity accelerates the onset of T1D (i.e. earlier or at a later stage in the natural history of the autoimmune process after substantial destruction of β cells has taken place) [50]. This controversy is particularly relevant for DD as these subjects are persistently obese and insulin resistant whereas the immunological markers of T1D represent an additional co-existent factor. To summarize, the greater the BMI and associated insulin resistance, the greater the damage to β cells in T1D, similar to what occurs for T2D. Follow-up studies are definitely necessary to sustain the accelerator hypothesis in DD.

Role of Autoimmunity to Beta Cells in Double Diabetes

Evaluation of autoantibodies to islet cell antigens and loss of C-peptide secretion is currently used for the prediction of T1D. Several studies have confirmed that the combination of three autoantibodies (GAD65, IA-2, and IAA) predicts T1D within 5 years in nearly all cases [51, 52]. One autoantibody alone confers low risk for progression; its positive predictive value should be calculated together with the occurrence of high risk human leukocyte antigens (HLA) genotypes as well as with the presence of a first-degree relative affected by the disease. Autoantibody affinity together with epitope analysis and their association with inter- and intra-molecular epitope spreading may help to refine the predictive value [53–55]. Thus, most GAD antibody-positive sera at the time of T1D diagnosis recognize conformationdependent epitopes located in the middle and the C-terminus of the GAD molecule, whereas few recognize the N-terminus. The appearance of GAD antibodies specific for conformational epitopes has been demonstrated longitudinally in individuals at risk of progression to T1D. The increase of GAD binding to the T1D-associated middle epitopes was observed in 72% of the high-risk children (of whom >40% developed diabetes during the follow-up period), whereas only 10% of children who do not progress to T1D showed this dynamic epitope change, which means a positive predictive value of 80% [55]. GAD antibodies can possess therefore a prognostic value based on their affinity and epitope analysis for determining an individual’s risk for progression to T1D. This rule applies for prediction of T1D as DD data have shown the presence of one single autoantibody, implying that progression to β cell destruction may be slower [56] although this has not been demonstrated. More

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studies are required to determine whether epitope change and its prognostic significance follows the same pattern in subjects with DD as it does in lean prediabetic individuals.

Diagnosis of Double Diabetes

If a teenager or child with all the typical clinical features of T2D, excess body weight, acanthosis nigricans, high blood pressure, high cholesterol, polycystic ovary syndrome (PCOS) (for girls), positive family history of T2D, belonging to an ethnic/ racial minority group, and show autoantibodies against insulin-producing β cells, we would say he/she has elements of both kinds of diabetes. A number of reports have described that as many as 15–20% of teens with the typical symptoms of T2D have autoantibodies circulating in their blood. Because of the presence of these autoantibodies, they can no longer be considered a pure T2D case. The following clinical and biochemical parameters can pave the way to diagnose a youth with DD: 1 Presence of clinical features of T2D, hypertension, dyslipidemia, increased BMI with increased cardiovascular risk compared with children with classical T1D. Family history for T2D and T1D may be present. 2 Presence of reduced number of clinical features typical of T1D such weight loss, polyuria/polydipsia, insurgence of ketoacidosis; insulin therapy is not the first line of therapy compared with subjects with classical T1D. 3 Presence of autoantibodies to islet cells but reduced number and titre compared to T1D, and probably reduced risk at HLA locus compared with subjects with T1D. As compared with T1D where insulin resistance and obesity are not common features, DD is always characterized by an obese phenotype with the addition of coexistent β cell autoimmunity.

Prevention

While relatively little is known about the long-term outcome of DD since it is a newly identified condition, we do know that being overweight and having diabetes genes in one’s family are the main causes. Since we cannot do anything about our genes, managing weight is the most important way to prevent DD. To prevent the development of DD, it is important to be physically active and prevent excessive weight gain or obesity. A healthy lifestyle that includes good eating habits and lots of physical activity benefits all children and adults regardless of their risk for diabetes. For a person with T1D that shows signs of DD, achieving and maintaining a healthy weight may resolve their body’s problem with insulin resistance (the basic feature of T2D and obesity). As weight decreases, the amount of insulin required to control glucose levels decreases

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and blood pressure and lipid levels also return to more normal (or optimal) levels. In women and girls, the polycystic ovary syndrome may also improve. Since DD is a new problem, there is not enough experience to decide what treatment approaches are most effective. There are a number of research studies underway which will provide guidance within the next few years. In the meantime, maintaining glucose control, achieving weight loss, being physically active and eating a nutritious diet are essential to managing DD and decreasing complications [57].

Therapy of Double Diabetes

Given the rapidly increasing problems associated with obesity, attempts to prevent the development of a condition such as DD may be highly relevant. Considering that in DD consistent β cell function is still present at the time of diabetes diagnosis and its decline may be slower than in classical T1D, an intervention capable of interfering with some of the putative mechanisms involved in the disease process may be worthwhile. Therefore, an immunomodulatory trial similar to one of those considered for T1D might be considered in DD. Although prevention of T1D is still far from being achieved, prevention of T2D has been shown to be feasible. Changes in lifestyle are relevant in halting or delaying the progression to overt disease in subjects at risk for T2D, probably as a consequence of an improvement in insulin sensitivity, as shown in the Diabetes Prevention Program [58]. Despite the increased prevalence of T2D in the pediatric population, there is limited information about the relative effectiveness of any treatment approach and pediatric diabetologists have had to rely on treatment paradigms derived from research and experience in the care of adults with T2D. Treatment Options for T2D in Adolescents and Youth (TODAY) is a randomized parallel group trial designed to test the hypothesis that aggressive reduction in insulin resistance early in the course of T2D is beneficial for prolongation of glycemic control, as well as improvement in associated abnormalities and risk factors [59]. Participants meeting eligibility criteria at the end of run-in are randomized to (1) metformin alone, (2) metformin plus rosiglitazone, or (3) metformin plus an intensive lifestyle intervention called TODAY Lifestyle Program. TODAY is the first large-scale, systematic study of treatment effectiveness for T2D in youth [59]. Whether such an approach may be successful in DD where, in addition, autoimmune phenomena also play a major role is unknown. New trials aimed at reducing insulin resistance using metformin and glitazones have been proposed for adult diabetic subjects with autoantibodies to β cells (LADA) to prevent the decline of β cell function [60, 61]. A treatment capable of interfering with the putative mechanisms involved in the disease process should be considered. In DD, a consistent β cell function is still present at the time of diagnosis, the decline of β cell function may be slower than in classical T1D and insulin resistance is present.

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Diet and Physical Activity Diet and physical exercise regimens have shown significantly greater effect in preventing the onset of T2D compared to placebo (58% decrease in incidence), and changes in lifestyle were even superior to metformin therapy (31% decrease in incidence) [62]. Whether such an approach may be successful in DD where autoimmune phenomena also play a major role is unknown Metformin Adjunctive metformin in overweight young people with T1D improves glycemic control without the weight gain expected with insulin therapy. In the long term, insulin treatment was associated with a fall in BMI [63]. Insulin In the Tokyo Study, 54 GAD-positive patients were randomly divided into 2 groups: one group received insulin (Ins group), the other a sulfonylurea (SU group). The study showed that small doses of insulin effectively prevent β cell failure in slowly progressive T1D, specifically in those patients with preserved β cell function and a high GAD titer at the initiation of therapy [64]. Other pharmacological potential drugs for treatment of DD include orlistat, sibutramine, glucosidase inhibitors, thiazolidinediones and glucagon-like peptide-1 [65–68].

Conclusions

Over the past decade, it has become apparent that more cases of T1D are diagnosed in children and adolescents who were overweight or even obese before hyperglycemia has developed. Sometime even diagnosis of T1D is not easy to place because of the phenotypic features typically associated with T2D. In addition, the increase of obesity observed in children may contribute to speed up the process of β cell destruction in subjects genetically susceptible to T1D. It is therefore necessary to investigate this new emerging form of DD in childhood and adolescence because it contributes overall to the increasing incidence of T1D amongst different populations. It is very likely that with increasingly greater effects of the environment on the onset of autoimmune disease – particularly in the group of subjects with DD – lifestyle modifications, including diet and exercise, may be relevant for the prevention of T1D in some populations, just as they are for the prevention of T2D. In conclusion, all forms of diabetes have shown a rapid increase over the past decade associated with a concomitant increase of overweight and obesity as well an earlier age of onset of the disease. Additionally, new diabetes phenotypes appear (DD) which render difficult both diagnosis and consequently the therapeutic approach to these patients who are mostly in the pediatric age group. Urgent action is needed to reverse this trend.

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Prof. Paolo Pozzilli Department of Endocrinolgy and Diabetes University Campus Bio-Medico, Via Alvaro del Portillo, 21 IT–00128 Rome (Italy) Tel. +39 06 22541 9184, Fax +39 06 22541 456, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 167–173

Cryptorchidism as Part of the Testicular Dysgenesis Syndrome: The Environmental Connection K.M. Maina ⭈ N.E. Skakkebæka ⭈ J. Topparib a

University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark; Departments of Physiology and Paediatrics, University of Turku, Turku, Finland

b

Abstract Cryptorchidism is part of the testicular dysgenesis syndrome (TDS), which includes other male reproductive disorders such as hypospadias, testis cancer and reduced semen quality. These diseases appear to be linked by common pathogenic mechanisms, interfering with normal fetal testis development. Testis development and descent is dependent on androgens and thus on an intact hypothalamus-pituitary-gonadal axis. Although cryptorchidism occurs in rare syndromes and genetic disorders, in the majority of children the etiology remains open. Many maternal and fetal risk factors have been previously identified but recently, scientific focus has also been directed to environmental hormone disrupting chemicals and lifestyle, as the prevalence of testis cancer and cryptorchidism has increased and semen quality decreased over few decades in several countries. Some persistent environmental chemicals, e.g. polychlorinated pesticides and polybrominated flame retardants, were associated with testicular maldescent and testis cancer. In addition, prenatal exposure to phthalates was negatively correlated to testosterone levels and anogenital distance as a measure of androgen effect in infant boys. Alcohol consumption and maternal smoking during pregnancy also appeared to be a risk factor for cryptorchidism. Thus, current evidence suggests that the development of the male reproductive tract may be susceptible to adverse effects of environmental horCopyright © 2009 S. Karger AG, Basel mone disrupters.

Testicular development and descent is regulated by numerous genetic factors, and is dependent on hormones produced by the Leydig cells, e.g. insulin-like factor 3 (Insl3) and testosterone [1]. Although several complex syndromes and genetic disorders are linked with testicular maldescent and other genital malformations [1], in the majority of boys with cryptorchidism no distinct etiology can be ascertained. Cryptorchidism is one of the most frequent congenital malformations in newborn boys. The reported prevalence of approximately 2–9% for congenital cryptorchidism differs considerably between studies and varies depending on diagnostic criteria, age

at examination, selection of study population, country of origin, ethnicity, as well as the experience of the observer [1]. Some countries, e.g. Great Britain and Denmark, have observed an increase in the prevalence of cryptorchidism over three to four decades [2], suggesting that environmental factors may play a role. A synchronous increase in the prevalence of male reproductive disorders over recent decades has been reported for hypospadias [3–5] and testicular cancer in several areas [6]. Semen quality appears to decline in many Western countries [7, 8]. These disorders have common pathogenic risk factors, they can present in combination and they are also risk factors for each other. Thus, we brought forward the hypothesis suggesting that male reproductive disorders were interlinked in a testicular dysgenesis syndrome (TDS) that originates from fetal life [9]. Many risk factors for cryptorchidism have been previously described and encompass paternal, maternal and fetal characteristics, e.g. intra-uterine growth restriction, low birth weight, prematurity, placental insufficiency, pre-eclampsia, maternal diabetes and siblings with cryptorchidism [1]. However, recent studies have highlighted the potential adverse role of environmental chemicals with hormone disrupting properties and lifestyle factors on fetal testicular development and subsequent male reproductive disorders [10].

Trends in the Prevalence of Male Reproductive Disorders

Registry data for congenital malformations such as cryptorchidism and hypospadias are of limited value to study time trends, as there are considerable variations in the degree of reporting and ascertainment [1]. Thus, only few prospective population-based studies are available, which document an increase in the prevalence of cryptorchidism [2] and hypospadias [3–5]. At the same time, there appears to be a decrease in semen quality parameters in many Western countries [7]. Testicular germ cell cancer, in turn, has been thoroughly registered in many countries and shows a considerable increase over the past few decades in many countries [6]. Genetic factors alone cannot explain such an increase, but this observation points towards other environmental factors or an influence by lifestyle. There is now considerable scientific evidence that testis cancer originates from carcinoma in situ testis, which is linked to prenatal dysgenetic development of the primordial germ cells in the testis [11]. Thus, an increase in testis cancer may be a general indicator of male reproductive health in a population [12].

Environmental Factors and Lifestyle

Chemical compounds with estrogenic or anti-androgenic properties can adversely affect the development of the testis [13]. Chemicals with endocrine disrupting

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properties are ubiquitously found in the environment and exposure routes are many, e.g. diet, air, cosmetics, cleaning substances, electronic equipment [14]. It is difficult to establish causal links between exposure to environmental chemicals and adverse outcomes in humans, as the human exposure situation is complex and interlinked with lifestyle factors and genetic susceptibility. Some chemical substances such as halogenated compounds bio-accumulate and are therefore present for many generations, whereas others, e.g. phthalates, appear to be rapidly metabolized. Thus, human evidence is usually derived from epidemiological studies or nested case control investigations within larger populations. These studies are laborious to perform and chemical analyses of trace amounts of environmental chemicals are expensive. It has often been experiences from wildlife and animal experiments that have given rise to concern about human health issues. In addition, toxicological studies in animals provide crucial information on mechanisms of hormone disruption. Thus, many scientific disciplines supplement each other and need to collaborate, in order to strengthen research in this area. Exposure to phthalates and their metabolites is an example, where interdisciplinary cooperation contributed to the discovery that the development of the foetal testis can be adversely affected by perinatal exposure. Although phthalates are rapidly metabolized, the exposure to them is ubiquitous and phthalate metabolites have thus been found in many biological fluids, including amniotic fluid and breast milk [15]. Prenatal exposure to phthalates was related to a reduced anogenital distance in boys, which in turn correlated with an increased prevalence of congenital cryptorchidism, smaller scrotal size and smaller penis volume [16]. Another study showed that the concentration of phthalate metabolites in breast milk was negatively correlated to serum free testosterone levels in 3-month-old boys [17]. In rats, in utero exposure to dibutyl phthalate causes a picture similar to that of TDS in man, especially if exposure was during the most sensitive developmental window [18]. This effect included cryptorchidism, hypospadias, decreased sperm count and decreased testosterone and Insl-3 production. Several modes of actions for phthalates and their metabolites have been proposed [15], but the predominant effect of phthalates on the developing testis appears to be a reduction in testosterone biosynthesis. Exposure to phthalate esters appeared to reduce the expression of genes involved in cholesterol transport and steroidogenesis and the availability of cholesterol for steroidogenesis. In epidemiological studies, associations have been reported between parental occupations in agricultural work with higher than average exposure to pesticides and a higher prevalence of cryptorchidism, and in part also hypospadias, in their male offspring. There is a higher rate of orchidopexies in areas of high pesticide use versus unexposed regions [19]. Few studies are available which employ individual measurements of pesticide levels in a biological matrix. Fat tissue samples from boys during operation for cryptorchidism showed higher concentrations of heptachloroepoxide and hexachlorobenzene than controls [20]. Some nested case-controls studies have found significant associations between the level of exposure to pesticides [19, 21]

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and polybrominated flame retardants [22] and congenital cryptorchidism. Although many of the investigated substances have been banned for use in Western countries, their persistency in the food chain may still contribute to the overall cocktail exposure of current populations. A very recent study investigated children of mothers who had worked in greenhouses before pregnancy and during the first weeks of gestation. This study could not investigate which of the many pesticides used during the study period was responsible for the detected effects. It showed, however, a threefold increase of cryptorchidism in the exposed sons and several indicators of reduced reproductive function such as reduced penile length, serum testosterone and an increased gonadotropin drive in the 3-month-old children [23]. Maternal exposure to persistent environmental chemicals, including polybrominated flame retardants, could also be linked to the development of testicular cancer in their sons two decades later [24]. The mechanisms of these adverse effects are yet unknown. Flame retardants may exert their effect in combination with simultaneous exposure to other chemicals and lifestyle factors. Recently, a new approach to toxicological testing has been developed to address so-called ‘cocktail effects’ of combined chemical exposures in rodents. In one study, the authors used three different anti-androgenic chemicals in a combined oral exposure, which led to additive adverse effects, e.g. nipple retention and reduced anogenital distance in the outcome [25]. The most striking evidence from these studies, however, was the observation that the combined exposure at dose levels which for the individual chemical were below any observed adverse effect level, resulted in a high prevalence of hypospadias in these animals [26]. These results are of particular concern for humans as the study concept reflects more realistically the human exposure situation than traditional toxicological experiments. From fetal life until adulthood, a human is simultaneously exposed to many chemicals at low levels, many of which accumulate over the lifetime. It is yet unknown how genetic susceptibility, including regulating factors of metabolic processes, may in addition influence the individual outcome of exposures. The general increase in the prevalence of obesity among Western populations may contribute to reproductive health problems. Thus, it has been shown that both under and overweight in young men was associated with reduced semen quality [27]. Mild gestational diabetes and even impaired glucose tolerance test represented a risk for congenital cryptorchidism [28]. This may be of considerable importance for public health as the prevalence of maternal pre-pregnancy obesity and thus gestational diabetes is increasing in many Western countries. There are also indicators that suggest an association between maternal lifestyle during pregnancy such as moderate regular maternal alcohol consumption and mild disorders of testicular descent [29]. Another study found a correlation between persistent cryptorchidism and binge drinking [30], but not regular alcohol intake. Previous retrospective and registry-based studies did not find these associations, which may be due to underreporting of this malformation and variation in the ascertainment of

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cryptorchidism. The precise mechanisms by which alcohol intake may affect testicular development in humans are currently unknown. Also, maternal smoking during pregnancy was found to be an independent risk factor for bilateral versus unilateral congenital cryptorchidism [31] and reduced semen quality in adult offspring [27, 32]. As the Danish and Finnish women differed considerably in their alcohol consumption patterns, this lifestyle difference may contribute to the difference seen in the prevalence of cryptorchidism in the two Nordic countries [2]. Congenital and adult male reproductive disorders are interlinked in the testicular dysgenesis syndrome, which suggests common pathogenic factors acting in utero. Several chemicals with known endocrine disrupting effects in animal experiments or in vitro are also associated with an increased risk of congenital cryptorchidism in humans or with signs of subtle impairment of testicular function. There is emerging evidence that some chemicals may be associated with testis cancer development. In addition, modern lifestyle factors such as obesity and gestational diabetes, maternal smoking and alcohol consumption during pregnancy appear to constitute independent risk factors for gonadal descent and function in the male offspring. As human exposure is complex, research in this field should be considerably strengthened in order to provide more insight into the mechanisms of how the different factors are interlinked. In addition, genetic polymorphisms may alter the extent to which adverse factors cause a specific phenotype.

Acknowledgements This work was supported by the Novo Nordisk Foundation, The European Commission, The Danish Medical Research Council, The Novo Nordisk Foundation, The Turku University Central Hospital, The Academy of Finland and the Sigrid Jusélius Foundation.

References 1

2

3

Virtanen HE, Toppari J: Epidemiology and pathogenesis of cryptorchidism. Human Reprod Update 2008;14:49–58. Boisen K, Kaleva M, Main KM, Virtanen HE, Haavisto A-M, Schmidt IM, Chellakooty M, Damgaard IN, Mau C, Reunanen M, Skakkebæk NE, Toppari J: Difference in the prevalence of congenital cryptorchidism in infants between two Nordic countries, Lancet 2004;363:1264–1269. Boisen KA, Chellakooty M, Schmidt IM, Kai CM, Damgaard IN, Suomi AM, Toppari J, Skakkebæk NE, Main KM: Hypospadias in a cohort of 1072 Danish newborn boys: prevalence and relationship to placental weight, anthropometrical measurements at birth and reproductive hormone levels at 3 months of age. J Clin Endocrinol Metab 2005;90:4041–4046.

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Paulozzi LJ, Erickson JD, Jackson RJ: Hypospadias trends in two US surveillance systems. Pediatrics 1997;100:831–834. Pierik FH, Burdorf A, Nijman JM, de Muinck Keizer-Schrama SM, Juttmann RE, Weber RF: A high hypospadias rate in the Netherlands. Hum Reprod 2002;17:1112–1115. Purdue MP, Devesa SS, Sigurdson AJ, McGlynn KA: International patterns and trends in testis cancer incidence. Int J Cancer 2005;115:822–827. Swan SH, Elkin EP, Fenster L: The question of declining sperm density revisited: an analysis of 101 studies published 1934–1996. Environ Health Perspect 2000;108:961–966.

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8 Jørgensen N, Asklund C, Carlsen E, Skakkebæk NE: Coordinated European investigations of semen quality: results from studies of Scandinavian young man are a matter of concern. Int J Androl 2006;29: 54–61. 9 Skakkebæk NE, Raipert-de Meyts E, Main KM: Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–978. 10 Skakkebæk NE, Jørgensen N, Main KM, Rajpert-De Meyts E, Lefferes H, Andersson AM, Juul A, Carlsen E, Mortensen GK, Jensen TK, Toppari J: Is human fecundity declining? Int J Androl 2006;29:2–11. 11 Rajpert-De Meyts E: Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update 2006;12:303–323. 12 Skakkebæk E, Rajpert-de Meyts E, Jørgensen N, Main KM, Leffers H, Andersson AM, Juul A, Jensen TK, Toppari J: Testicular cancer trends as ‘whistle blowers’ of testicular developmental problems in populations. Int J Androl 2007;30:198–2005. 13 Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ Jr, Jégou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Müller JR, Rajpert-De Meyts E, Scheike T, Sharpe RM, Sumpter J, Skakkebæk NE: Male reproductive health and environmental xenoestrogens. Environ Health Perspect 1996;104: 741–803. 14 Damgaard IN, Main KM, Toppari J, Skakkebæk NE: Impact of exposure to endocrine disrupters in utero and in childhood on adult reproduction: best practice and research, clinical endocrinology and metabolism. Baillières Endocrinol 2002;16:289– 309. 15 Lottrup G, Andersson AM, Leffers H, Mortensen GK, Toppari J, Skakkebæk NE, Main KM: Possible impact of phthalates on infant reproductive health. Int J Andrology 2006;29:172–180. 16 Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon JB, Ternand CL, Sullivan S, Teague JL, Study for Future Families Research Team: Anogenital distance – a marker of fetal androgen action – is decreased in male infants following phthalate exposure during pregnancy. Environ Health Perspect 2005;113:1056–1061. 17 Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi AM, Virtanen HE, Petersen JH, Andersson AM, Toppari J, Skakkebæk NE: Human breast milk contamination with phthalates and alterations in endogenous reproductive hormones in three months old children. Environ Health Perspect 2006;114:270–276.

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18 Welsh M, Saunders PT, Fisken M, Scott HM, Hutchinson GR, Smith LB, Sharpe RM: Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 2008; 118:1479–1490. 19 Damgaard IN, Skakkebæk NE, Toppri J, Virtanen HE, Shen H, Schramm KW, Petersen JH, Jensen TK, The Nordic Cryptorchidism Study Group, Main KM: Persistent pesticides in human breast milk and cryptorchidism. Environ Health Perspect 2006;114: 1133–1138. 20 Hosie S, Loff S, Witt K, Niessen K, Waag KL: Is there a correlation between organochlorine compounds and undescended testes? Eur J Pediatr Surg 2000;10: 304–309. 21 Fernandez MF, Olmos B, Granada A, Lopez-Espinosa MJ, Molina-Molina JM, Fernandez JM, Cruz M, OleaSerrano F, Olea N: Human exposure to endocrinedisrupting chemicals and prenatal risk factors for cryptorchidism and hypospadias: a nested case-control study. Environ Health Perspect 2007;115:8–14 22 Main KM, Kiviranta H, Virtanen HE, Sundqvist E, Tuomisto JT, Tuomisto J, Vartiainen T, Skakkebæk NE, Toppari J: Flame retardants in placenta and breast milk and cryptorchidism in newborn boys. Environ Health Perspect 2007;115:1519–1526 23 Andersen HR, Schmidt IM, Grandjean P, Jensen TK, Budtz-Jørgensen E, Kjærstad MB, Bælum J, Nielsen JB, Skakkebæk NE, Main KM: Impaired reproductive development in sons of women occupationally exposed to pesticides during pregnancy. Environ Health Perspect 2008;116:566–557. 24 Hardell L, Van Bavel B, Lindstrom G, Eriksson M, Carlberg M: In utero exposure to persistent organic pollutants in relation to testicular cancer risk. Int J Androl 2006;29:229–234. 25 Hass U, Scholze M, Christiansen S, Dalgaard M, Vingaard AM, Axelstad M, Metzdorff SB, Kortenkamp A: Combined exposure to anti-androgens exacerbates disruption of sexual differentiation in the rat. Environ Health Perspect 2007;115: 122–128. 26 Christiansen S, Scholze M, Axelstad M, Boberg J, Kortenkamp A, Hass U: Combined exposure to anti-androgens causes markedly increased frequencies of hypospadias in the rat. Int J Androl 2008; 1:241–248. 27 Jensen TK, Jørgensen N, Punab M, Haugen TB, Suominen J. Zilaitiene B, Horte A, Andersen AG, Carlsen E, Magnus Ø, Matulevicius V, Nermoen I, Vierula M, Keiding N, Toppari J, Skakkebæk NE: Association of in utero exposure to maternal smoking with reduced semen quality and testis size in adulthood: a cross-sectional study of 1770 young men from the general population in five European countries. Am J Epidemiol 2004;159:49–58.

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28 Virtanen HE, Tapanainen AE, Kaleva MM, Suomi AM, Main KM, Skakkebæk NE, Toppari J: Mild gestational diabetes as a risk factor for congenital cryptorchidism. J Clin Endocrinol Metab 2006;91: 4862–4865. 29 Damgaard IN, Jensen TK, the Nordic Cryptorchidism Study Group, Petersen JH, Skakkebæk NE, Toppari J, Main KM: Cryptorchidism and maternal alcohol consumption during pregnancy. Environ Health Perspect 2007;115:272–277 30 Jensen MS, Bonde JP, Olsen J. Prenatal alcohol exposure and cryptorchidism. Acta Paed 2007;96: 1681–1685.

31 Thorup J, Cortes D, Petersen BL: The incidence of bilateral cryptorchidism is increased and the fertility potential is reduced in sons of mothers who have smoked during pregnancy. J Urol 2006;176:734– 737. 32 Storgaard L, Bonde JP, Ernst E, Spanò M, Andersen CY, Frydenberg M, Olsen J: Does smoking during pregnancy affect sons’ sperm counts? Epidemiology 2003;14:278–286.

Katharina M. Main MD, PhD University Department of Growth and Reproduction Section 5064, Rigshospitalet Blegdamsvej 9, DK--2100 Copenhagen (Denmark) Tel. +45 3545 5085, Fax +45 3545 6054, E-Mail [email protected]

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Cappa M, Maghnie M, Loche S, Bottazzo GF (eds): Endocrine Involvement in Developmental Syndromes. Endocr Dev. Basel, Karger, 2009, vol 14, pp 174–180

Disorders of Sex Development in Developmental Syndromes Olaf Hiorta ⭈ Gabriele Gillessen-Kaesbachb a

Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, and bInstitut für Humangenetik, Universität zu Lübeck, Germany

Abstract Disorders of sex development comprise an array of congenital conditions with atypical development of chromosomal, gonadal, and anatomical sex affecting the genitourinary tract and in most instances also the endocrine-reproductive system. While the molecular basis of some of these disorders has been well established, it remains elusive in others. This holds true especially for disorders of sex development that are associated with other congenital malformations and abnormalities in a ‘syndromic’ condition. Syndromic disorders of sex development may be due to monogenic defects, biochemical abnormalities of steroid synthesis, or cytogenetic abnormalities comprising microdeletions or duplications or unbalanced rearrangements. This review will focus on the clinical description of syndromic disorders of sex development guiding towards the genetic classification. The characterization of the underlying diagnosis will improve genetic counseling of the family including progCopyright © 2009 S. Karger AG, Basel nosis and recurrence risk.

‘Disorders of sex development (DSD)’ comprise a heterogeneous group of heritable disorders of sex determination and differentiation, formerly termed ‘intersexuality’. This includes chromosomal as well as monogenic disorders, which inhibit or change primarily genetic or endocrine pathways of normal sex development. The elucidation of the underlying cause may be extremely helpful in counseling patients and families with respect to gender assignment, overall prognosis and establishing an individualized management of the patient [1].

Interdisciplinary Approach to Syndromic DSD

In a subset of patients, DSD is part of a syndrome with other associated congenital malformations, Biochemical and genetic studies have elucidated common pathways and have identified the causative genes in a variety of DSD syndromes. In other

conditions, the etiology is yet unclear. It is very important to care for these patients in an interdisciplinary team consisting of pediatric surgeons/urologists, human geneticists, and pediatric endocrinologists. [2]. It is their task to develop diagnostic strategies helping to define the underlying genetic or biochemical disorder to allow for a stepwise and stringent management of the patient. In order to classify the underlying disorder, a detailed clinical examination and description, a biochemical age-related referenced endocrine profile, as well as genetic investigations are necessary. Clinical examination should reveal all abnormalities seen in the patient and include auxological measurements. It is very important to receive a detailed family history as well as precise data on the past medical history of the patient including birth data. In addition, imaging investigation by ultrasound of the morphological abnormalities of the urogenital tract are the best and fastest ways for an initial description and classification. A supplementary description of the anatomy and the type of gonadal abnormality may be needed, involving a pediatric surgeon’s description of the inner genitalia, namely the status of Wolffian duct or Mullerian duct development as well as the histological investigation of the gonads. During the newborn period and early infancy, endocrine investigations may be directed by the endogenous stimulation of the ‘mini-puberty’ to describe adrenal and gonadal disorders. Serum analysis can be complemented by a urinary steroid profile involving gas chromatography/ mass spectrometry, which will allow for identification of various adrenal abnormalities. Any biochemical analysis during infancy and childhood needs to be performed in a specialized pediatric-endocrine laboratory that has the special attire needed for these analyses and uses the respective age-related reference values. Cytogenetic analysis complemented by CGH array analysis may help to identify the underlying genetic defect. The results of this diagnostic approach may then allow classification of the syndromic disorder either as a distinct entity of monogenic origin, caused by a developmental gene defect or a biochemical abnormality as well caused by a single gene mutation.

Distinct Monogenic Syndromic Developmental Disorders with DSD

The initial, sexually indifferent phase of gonad formation begins at 5 weeks of gestation with the development of paired gonadal ridges. The process becomes sexually dimorphic after germ cell seeding at 6 weeks and bipotential gonad formation at seven weeks. Those gonads with a 46,XY karyotype begin expressing SRY (sex-determining region on the Y chromosome), the transcription factor which is thought to initiate the downstream molecular events of testis formation [3]. These transcription factors involved in bipotential gonad formation are necessary for developmental processes in other tissues, and their disruption often is associated with nongonadal malformations and diseases. An increasing variety of clinical syndromes with ambiguous

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genitalia are now elucidated on the molecular level. In the following a few examples are described in more detail (table 1). Mutations in the Wilms’ tumor 1 (WT1) gene lead to several clinical entities [4]. In addition to disruption of gonadal development in both sexes, the differentiation of the kidneys can be disturbed, leading to the formation of Wilms’ tumors during childhood. Moreover, chromosomal deletions of 11p13, the location of the WT1 gene, have been associated with aniridia, Wilms’ tumor and mental retardation as part of the WAGR-syndrome. Point mutations in the WT1 gene lead to DenysDrash and Frasier syndromes. In Denys-Drash syndrome, Wilms’ tumors are associated with severe kidney disease with mesangial sclerosis and gonadal dysgenesis. In Frasier syndrome, a complete gonadal dysgenesis is accompanied by a late-onset focal glomerulosclerosis and a high risk for gonadoblastoma instead of Wilms’ tumors. Of note, an abnormality of the external genitalia is only present in children with a 46,XY karyotype, because only there diminished androgen synthesis will cause a visible malformation. However, the developmental defects will be present in both sexes. In contrast to WT1 mutations, genetic alterations in the steroidogenic factor 1 (SF1) encoded by the NR5A1 gene will lead to gonadal dysfunction of variable extent and may be associated with severe adrenal failure due to a developmental defect of adrenal steroidogenesis [5]. While this is overt in some cases at birth, adrenal failure may develop later in others and has to be kept in mind [6]. Another example of a single gene involved in formation of the bipotent gonad in association with other malformations is the hand-foot-genital syndrome caused by mutation of the homeobox gene on chromosome 7 designated HOXA13. Females have duplication of the uterus and sometimes of the cervix, and might have a septated vagina. In males hypospadias are characteristic. The hands are small, the thumbs are malformed and the thenar eminence is flat. In the feet the first toe is small, and radiographs show a short first metacarpal and phalanx. Shortly after SRY expression, the SRY-related HMG-Box gene 9 (SOX9) gene, which is required for Sertoli cell differentiation and type II collagen production, begins to be expressed in the testes [3]. Patients with mutations in the SOX9 gene show bowing of the femur and tibia, a large head, a small jaw, a cleft palate and a flat nasal bridge. Congenital dislocation of the hip and heart defects are common. Characteristically, the scapulae are hypoplastic and the iliac wings are vertical and narrow. Ambiguous genitalia occur in the majority of patients with a 46,XY karyotype. A duplication of SOX9 can lead to a testis formation in individuals with a 46,XX karyotype, demonstrating the importance of gene dosis effects in gonadal differentiation. Other transcription factors like FGFR2 (10q26) have been associated with severe genital malformations in animals or humans. Mutations in the Fgfr2 gene lead to partial sex reversal in male mice; loss of the terminal 10q26 region also has been associated in humans with sex reversal, but a specific association with mutations in the Fgfr2 gene has not been described in humans. Similarly, loss of Fgf9, an inducer of Fgfr2, leads to disorders of sex development in male vertebrates but has not yet been

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Table 1. Genes known to be involved in syndromal forms of disorders of sex development Gene

Locus

Mullerian structures

External genitalia

Associated features

46,XY DSD single gene disorders of gonadal development WT1 11p13 AD dysgenetic

+/–

ambiguous to female

SF1

9q33

AD/AR

dysgenetic

+/–

SOX9

17q24-25

AD

+/–

ARX

Xp22.13

XR

dysgenetic or ovotestis dysgenetic

ambiguous to female ambiguous to female ambiguous

Wilms’ tumor, renal abnormalities, gonadal tumors Primary adrenal insufficiency Campomelic dysplasia

ATRX

Xq13.3

XR

dysgenetic

–

HOXA13

7p15

AD

unknown

–

Ambiguous to female Hypospadias

46,XX DSD single gene disorders of gonadal development RSPO1 1p34.3 AR ovotestis

+

ambiguous

WNT-4

–

male

1p35

Inheritance

AR

Gonad

testis

–

46,XY DSD with gene rearrangements, disorders of gonadal development DAX1 Xp21.3 duplication dysgenetic +/– ambiguous to female DMRT1+2 9p24.3 deletion dysgenetic +/– ambiguous to female WNT4 1p35 duplication dysgenetic +/– ambiguous to female

X-linked lissencephaly, epilepsy, temperature instability α-Thalassaemia, mental retardation Hand and foot abnormalities Keratoderma, hearing impairment, corneal opacities Renal, adrenal and pulmonary defects

Mental retardation, renal abnormalities, Mental retardation

46,XX DSD with gene rearrangements, disorders of gonadal development SOX9 17q24-25 duplication unknown – ambiguous to male SRY Yp11.3 translocation testis – ambiguous to female 46,XY DSD disorders of steroid synthesis with associated malformations DHCR7 11q12-13 AR testis –

POR

7q11.2

AR

testis

–

46,XX DSD disorders of steroid synthesis with associated malformations POR 7q11.d AR ovary +

Syndromic DSD

variable ambiguous

male to ambiguous ambiguous

Smith-Lemli-Opitz syndrome with facial abnormalities, clinoand syndactyly, other defect, mental retardation Antley-Bixler syndrome with craniosynostosis Antley-Bixler syndrome with craniosynostosis

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reported in humans. Instead, mutations in these genes can induce various skeletal dysplasias. The association of mental retardation and anomalies of the brain with genital malformations affecting children with a 46, XY karyotype has been reported for two different X-linked disorders, namely the α-thalassemia-mental retardation (ATRX) syndrome (ATRX gene on Xp13) and disorders showing mutations in the aristalessrelated homeobox gene (ARX gene on Xp22). The latter is characterized by a wide phenotypic spectrum including the infantile epileptic-dyskinetic encephalopathy (West syndrome) or a conditon characterized by lissencephaly, hypoplastic corpus callosum and ambiguous genitalia (XLAG syndrome). The association of palmoplantar keratoderma, congenital bilateral corneal opacities, onychodystrophy, hearing impairment and ovotesticular DSD in a child with 46,XX karyotype was found to be caused by a homozygous mutation in R-spondin 1 (RSPO1 gene), which also points out the pivotal role of this gene in gonadal differentiation due to regulation of the Wnt-4 pathway [7]. Wnt-4 and Wnt-7a are signaling molecules found in Müllerian ducts and show XX-specific gonadal expression. In Sertoli and Leydig cells, Wnt-4 overexpression upregulates the dose-sensitive sex-reversal locus on the X chromosome (DAX-1) and this may explain the etiology of human XY sex reversal associated with 1p35-p31 duplication. However, recently a novel autosomal-recessive syndrome (Serkal syndrome) was described that consists of female to male sex reversal and renal, adrenal, and lung dysgenesis and is associated with additional developmental defects. Using a candidate-gene approach, a disease-causing homozygous missense mutation in the human WNT4 gene was identified. The mutation was found to result in markedly reduced WNT4 mRNA levels in vivo and in vitro and to down regulate WNT4dependent inhibition of β-catenin degradation [8]. DAX1 is a gonad-specific transcription factor upregulated in the ovary. Mutations in DAX-1 are responsible for adrenal hypoplasia congenita (AHC), a syndrome of adrenal hypoplasia and hypogonadotropic hypogonadism in 46,XY individuals with an unequivocal male phenotype. DAX1 is said to function as an anti-testis factor in the ovary but is not required for normal testicular function. In contrast, DAX1 duplication can repress SRY and causes 46,XY sex reversal [9]. While the defects of testicular/ovarian-specific transcription factors, such as SRY or DAX-1, are not usually associated with other anomalies, it largely depends on the size and localization of the duplication /deletion that other genes may be also be compromised and, hence, the resulting phenotype is a complex syndrome. Single gene mutations have not been documented as the cause for DSD in the genes DMRT1 and DMRT2; instead the deletion of the genes is often due to distal monosomy of chromosome 9p. Patients with a 46,XY karyotype often have completely female external genitalia, however, also the presence of ovotesticular DSD has been described. A mental retardation of varying degree has been described. Additional described features include severe growth retardation [10].

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Hiort · Gillessen-Kaesbach

Syndromic DSD as a Consequence of Defects in Biochemical Pathways

Two distinct biochemical disorders have been described that present with supplementary malformations in addition to DSD. Antley-Bixler syndrome was initially described a as trapezoidocephaly synostosis syndrome caused by mutations in the fibroblast growth factor receptor gene FGFR2. However, an Antley-Bixler-like phenotype exists, where patients have additional ambiguous genitalia with both 46,XX and 46,XY karyotypes and biochemical features of a combined 21-hydroxylase (CYP21) and 17α-hydroxylase (CYP17) deficiency [11]. Although androgen levels may be low in infants with a 46,XX karyotype after birth, they may be severely virilized and even their mothers can virilize, which pointed to an alternative steroid metabolism during pregnancy. In these cases, Antley-Bixler syndrome is caused by inactivating mutations in P450-oxidoreductase (POR gene), which is a flavoprotein and an electron donor to all microsomal P450 enzymes. Smith-Lemli-Opitz syndrome (SLOS) is an autosomal-recessive disorder with multiple congenital malformations and mental retardation of variable expression. The patients may exhibit microcephaly, hypotonia, facial dysmorphism and ambiguous genitalia. Also, congenital heart defects as well as skeletal abnormalities are found. The latter often consist of cutaneous syndactyly of toes 2 and 3 and postaxial polydactyly. SLOS is due to mutations in the gene encoding for the cholesterol biosynthesis enzyme 7-dehydrocholesterol reductase [12]. Hence, plasma cholesterol may be low, and the substrate 7-dehydrocholesterol is elevated. The biochemical findings, however, do not correlate with the phenotypic expression and severity.

Unclassified Syndromic DSD

A variety of disorders of sex development remain unclassified today and their cause elusive. This includes distinct entities like the Muller-Rokitansky-Kuster-Hauser syndrome with vaginal and uterine aplasia in association with renal malformations in females with a 46,XX karyotype, but also the bladder exstrophy and epispadias complex as well as many other complex malformations involving the genito-urinary tract. In these cases, a distinction has to be made between developmental syndromes involving the morphologic formation of the external and internal genitalia secondary to an overruling malformation of the pelvic region and syndromes that may inhibit genital malformation secondary to gonadal or endocrine involvement as described above. In any case, a stringent and stepwise clinical, endocrine and genetic differential diagnosis has to be established. Employing modern molecular genetic techniques like ultra high resolution CGH, an attractive powerful approach can be offered that can detect minor changes in the genome which could contribute to or be responsible for the phenotype. Using these modern techniques, further syndromes involving the genitor-urinary tract will be elucidated in the near future.

Syndromic DSD

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References 1

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Hughes IA, Houk C, Ahmed SF, Lee PA: LWPES Consensus Group, ESPE Consensus Group: Consensus statement on management of intersex disorders. Arch Dis Child 2006;91:554–563. Hiort O, Holterhus PM: Molecular and hormone dependent events in sexual differentiation; in Henry A, Norman A (eds): Encyclopedia of Hormones. Los Angeles, Academic Press, 2004, pp 349–356. Clarkson MJ, Harley VR: Sex with two SOX on: SRY and SOX9 in testis development. Trends Endocrinol Metab 2002;13:106–111 Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L, Fine RN, Silverman BL, Haber DA, Housman D: Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 1991;67:437–447 Achermann JC, Ito M, Ito M, Hindmarsh PC, Jameson JL: A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet 1999;22:125–126. Köhler B, Lin L, Ferraz-de-Souza B, Wieacker P, Heidemann P, Schröder V, Biebermann H, Schnabel D, Grüters A, Achermann JC: Five novel mutations in steroidogenic factor 1 (SF1, NR5A1) in 46,XY patients with severe underandrogenization but without adrenal insufficiency. Hum Mutat 2008;29: 59–64

7 Tomaselli S, Megiorni F, De Bernardo C, Felici A, Marrocco G, Maggiulli G, Grammatico B, Remotti D, Saccucci P, Valentini F, Mazzilli MC, Majore S, Grammatico P: Syndromic true hermaphroditism due to an R-spondin1 (RSPO1) homozygous mutation. Hum Mutat 2008;29:220–226. 8 Mandel H, Shemer R, Borochowith ZU, Okopnik M, Knopf C, Indelman M, Drugan A, Tiosano D, Gershoni-Baruch R, Choder M, Sprecher E: SERKAL syndrome: an autosomal-recessive disorder caused by a loss-of-function mutation in WNT4. Am J Hum Genet 2008;82:39–47. 9 Barbaro M, Oscarson M, Schoumans J, Staaf J, Ivarsson SA, Wedell A: Isolated 46,XY gonadal dysgenesis in two sisters caused by a Xp21.2 interstitial duplication containing the DAX1 gene. J Clin Endocrinol Metab 2007;92:3305–3313. 10 Raymond CS, Parker ED, Kettlewell JR, Brown LG, Page DC, Kusz K, Jaruzelska J, Reinberg Y, Fletjer WL, Bardwell VJ, Hirsch B, Zarkower D: A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum Molec Genet 1999;8:989–996. 11 Arlt W, Walker EA, Draper N, Ivison HE, Ride JP, Hammer F, Chalder SM, Borucka-Mankiewicz M, Hauffa BP, Malunowicz EM, Stewart PM, Shackleton CH: Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study. Lancet 2004;363:2128– 2135. 12 Porter FD: Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management. Eur J Hum Genet 2008;16:535–541.

Prof. Dr. Olaf Hiort Division of Pediatric Endocrinology and Diabetes Department of Pediatrics, University of Lübeck Ratzeburger Allee 160, DE–23538 Lübeck (Germany) Tel. +49 451 500 2191, Fax +49 451 500 6867, E-Mail [email protected]

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Author Index

Accadia, M. 20 Ballarini, E. 114 Barberi, L. 29 Bergamaschi, R. 114 Bertini, E. 38 Bocciardi, R. 61 Calandra, E. 83 Camacho-Hübner, C. 143 Cerrato, F. 1 Chernausek, S.D. 135 Cicognani, A. 114 Citro, V. 1 Clark, A.J.L. 143 Cubellis, M.V. 1 D’Amico, A. 38 Dattani, M.T. 67 David, A. 143 De Crescenzo, A. 1 di Iorgi, N. 83 Dobrowolny, G. 29 Eggermann, T. 10 Ferrero, G.B. 1 Giacinti, C. 29 Gillessen-Kaesbach, G. 174 Guglielmi, C. 151 Gurrieri, F. 20 Hiort, O. 174 Hussain, K. 95

Kapoor, R.R. 95 Kelberman, D. 67 Larizza, L. 1 Maghnie, M. 83 Main, K.M. 167 Mazzanti, L. 114 Metherell, L.A. 143 Montanari, F. 114 Moscarda, M. 53 Musarò, A. 29 Napoli, F. 83 Neri, G. 53 Pelosi, L. 29 Pozzilli, P. 151 Ravazzolo, R. 61 Riccio, A. 1 Rossi, A. 83 Russo, S. 1 Savage, M.O. 143 Scarano, E. 114 Secco, A. 83 Silengo, M.C. 1 Skakkebæk, N.E. 167 Sparago, A. 1 Tamburrino, F. 114 Toppari, J. 167 Torella, M. 114 Verde, G. 1

James, C. 95

181

Subject Index

ABCC8, hyperinsulinemic hypoglycemia mutations 98, 99, 106 Achondroplasia clinical features 126 etiology 126 growth 126 growth hormone secretion and therapy 126, 127 Acid labile subunit (ALS), defects 147 Alpers-Huttenlocher syndrome, clinical features 47 Alpers syndrome clinical features 42 gene mutations 41 ALS, see Acid labile subunit Angelman syndrome (AS) clinical features 26 epidemiology 26 genetic counseling 27 imprinting regulation 22, 23, 26, 27 Antley-Bixler syndrome, gene mutations 179 AS, see Angelman syndrome ATRX, mutations in disorders of sexual development 178 Bannayan-Riley-Ruvalcaba syndrome (BRRS) clinical features 58 gene mutations 58 Barth syndrome, clinical features 48 BCS1L, mutation in mitochondrial diseases 46 Beckwith-Wiedemann syndrome (BWS) clinical features 2, 3, 53, 54, 97 epidemiology 53 gene mutations 3, 54, 55, 97, 98 hyperinsulinemic hypoglycemia 97–100 imprinting center 1 clinical effects of aberrations 4, 5

182

epigenetic mosaicism 5, 6 hypermethylation 4 IGF2-H19 imprinting loss 6, 7 microdeletions 3, 4 imprinting center 2 defects 98 BMI, see Body mass index Body mass index (BMI), obesity adults 153 children 153, 154 BRRS, see Bannayan-Riley-Ruvalcaba syndrome BWS, see Beckwith-Wiedemann syndrome CACNA1C, Timothy syndrome mutations 105 CDG, see Congenital defects of glycosylation CNP, see C-type natriuretic peptide Congenital defects of glycosylation (CDG), hyperinsulinemic hypoglycemia 96, 97, 106, 107 Costello syndrome gene mutations 102 hyperinsulinemic hypoglycemia 101, 102 COX, see Cyclooxygenase Cryptorchidism environmental and lifestyle risk factors 168–171 prevalence 167, 168 C-type natriuretic peptide (CNP) gene 63 mutations and stature 63 overexpression, growth aberrations, and mechanisms 63–66 signaling 62, 63 Cyclooxygenase (COX), deficiency 40, 41 DAX1, mutations in disorders of sexual development 178

de Toni-Debré-Fanconi syndrome, clinical features 43 DGUOK, mutation in mitochondrial diseases 47 Diabetes double diabetes beta cell autoimmunity 160, 161 diagnosis 161 environmental factors 159, 160 genes 159 latent autoimmune diabetes in the adult 158 prevalence in children 158, 159 prevention 161, 162 treatment 162, 163 obesity association 152, 153 type 1 versus type 2 151, 152 Disorders of sexual development (DSD) biochemical pathway defects and syndromic disorders 179 interdisciplinary approach to syndromic disorders 174, 175 monogenetic syndromic developmental disorders 175–178 unclassified syndromic disorders 179 Double diabetes, see Diabetes DSD, see Disorders of sexual development

growth 121 growth hormone secretion and therapy 121

Ectopic posterior pituitary (EPP) clinical features 85–87 endocrine consequences 90, 91 gene mutations 87–89 idiopathic forms 89, 90 stature implications 92 Ellis-van Crevald syndrome clinical features 129 etiology 129 growth 129 growth hormone secretion and therapy 129 Epigenetics, see also Imprinting reprogramming 21, 22 Silver-Russell syndrome chromosome 11 14, 15 imprinted gene networks 16, 17 EPP, see Ectopic posterior pituitary

Genomic imprinting, see Imprinting GH, see Growth hormone Glycosylation defects, see Congenital defects of glycosylation GPC3, Simpson-Golabi-Behmel syndrome mutations 55 Growth hormone (GH) insensitivity syndromes classification 143, 144 idiopathic short stature 147 insulin-like growth factor-I therapy adverse effects 140, 141 body composition effects 140 growth rates 137, 138 metabolic effects 139, 140 organ growth 138, 139 rationale 135, 136 Laron syndrome 136, 144, 145 receptor mutations dominant negative heterozygous receptor mutation 146 pseudo-exon receptor mutation 145 without Laron syndrome 145 STAT5b mutations 146, 147 treatment strategies 136, 137 secretion and therapy achondroplasia 126, 127 Ellis-van Crevald syndrome 129 Floating-Harbor syndrome 121 hypochondroplasia 128 Kabuki syndrome 122 Leri-Weill syndrome 130 Noonan syndrome 118 Pallister-Hall syndrome 123, 131 Prader-Willi syndrome 24, 25, 120, 131 pseudoachondroplasia 129 Silver-Russell syndrome 124, 125 Turner syndrome 116, 117 therapy benefits 132 complications 132 dosing 132 prospects for study 133

FGFR2, mutations in disorders of sexual development 176 Floating-Harbor syndrome clinical features 120, 121

H19, see Imprinting center 1 HESX1 ectopic posterior pituitary role 88 genotype-phenotype correlations 77

Subject Index

183

HESX1 (continued) hypopituitarism role 67 knockout mouse 85 septo-optic dysplasia mutations 68 HOXA13, mutations in disorders of sexual development 176 HRAS, Costello syndrome mutations 102 Hyperinsulinemic hypoglycemia Beckwith-Wiedemann syndrome 97–100 congenital defects of glycosylation 96, 97, 106, 107 Costello syndrome 101, 102 Kabuki syndrome 102, 103 overview of associated syndromes 96 Simpson-Golabi-Behmel syndrome 101 Sotos syndrome 100 Timothy syndrome 104, 105 transient versus persistent 95, 96 trisomy 13 103 Turner syndrome 103, 104 Usher syndrome 105, 106 Hypochondroplasia clinical features 127 etiology 127 growth 127 growth hormone secretion and therapy 128 IC1, see Imprinting center 1 Idiopathic short stature (ISS), management 147 IGF-1, see Insulin-like growth factor-1 IGF-2, see Insulin-like growth factor-2 Imprinting, see also Epigenetics Angelman syndrome 22, 23, 26, 27 epigenetic reprogramming 21, 22 overview 2, 20, 21 Prader-Willi syndrome 22–26 Imprinting center 1 (IC1) Beckwith-Wiedemann syndrome clinical effects of aberrations 4, 5 epigenetic mosaicism 5, 6 hypermethylation 4 IGF2-H19 imprinting loss 6, 7 microdeletions 3, 4 genes 2 Wilms’ tumor and absence of microdeletions 7, 8 Insulin double diabetes management 163 hyperinsulinemia, see Hyperinsulinemic hypoglycemia

184

Insulin-like growth factor-1 (IGF-1) acid labile subunit defects 147 gene defects 147, 148 gene structure 30, 31 isoforms clinical importance 32, 33 local effects on muscle homeostasis and regeneration 33, 34 transcription 31, 32 transgenic mouse studies of muscular dystrophy and muscle wasting 34, 35 resistance syndromes classification 143, 144 receptor defects 148 structure 30 supplementation rationale 34–36 therapy for growth hormone insensitivity syndromes adverse effects 140, 141 body composition effects 140 growth rates 137, 138 metabolic effects 139, 140 organ growth 138, 139 rationale 135, 136 transcription 30–32 Insulin-like growth factor-2 (IGF-2), see Imprinting center 1 ISS, see Idiopathic short stature Kabuki syndrome clinical features 102, 121 etiology 121 gene mutations 102 growth 121 growth hormone secretion and therapy 122 hyperinsulinemic hypoglycemia 102, 103 KCNJ11, hyperinsulinemic hypoglycemia mutations 98, 99 Kearn-Sayre syndrome (KSS) clinical features 42, 43, 45, 48 gene mutations 40 KSS, see Kearn-Sayre syndrome Laron syndrome, see Growth hormone Latent autoimmune diabetes in the adult, see Diabetes Leber’s hereditary optic neuropathy (LHON) clinical features 49 gene mutations 40 Leigh syndrome clinical features 42, 49, 50

Subject Index

gene mutations 40 Leri-Weill syndrome clinical features 129 etiology 130 growth 130 growth hormone secretion and therapy 130 LHON, see Leber’s hereditary optic neuropathy LHX3 genotype-phenotype correlations 77 hypopituitarism role 67, 74 structure 72, 73 LHX4 ectopic posterior pituitary role 89 genotype-phenotype correlations 77 hypopituitarism role 67, 73, 74 MDDS, clinical features 49 MELAS, clinical features 42, 43, 45 MERRF, see Myoclonus epilepsy and ragged red fibers Metformin, double diabetes management 162, 163 Mitochondrial diseases, see also specific diseases clinical presentation 39 diagnosis 39, 40 DNA abnormalities 38 genetics 40, 41 syndromes in cytopathies blood 48, 49 central nervous system 42, 43 endocrine system 45 gut 44 heart 44, 45 kidney 43, 44 liver 46–48 myopathy 41, 42 skin 49, 50 symptoms 42 MLASA, clinical features 48 MNGIE, clinical features 41, 44 Myoclonus epilepsy and ragged red fibers (MERRF), clinical features 42, 43, 49 Navajo neurohepatopathy (NNH), gene mutations 48 NNH, see Navajo neurohepatopathy Noonan syndrome clinical features 117 etiology 117 growth 118 growth hormone secretion and therapy 118

Subject Index

NR5A1, mutations in disorders of sexual development 176 NSD1, Sotos syndrome mutations 56, 100 Obesity consequences 158 definitions adults 153 children 153, 154 diabetes association 152, 153 etiology 156, 157 prevalence 153 trends in children 154–156 Pallister-Hall syndrome clinical features 122 etiology 122 growth hormone secretion and therapy 123, 131 Pallister-Killian syndrome (PKS) clinical features 59 prenatal diagnosis 59 Patau syndrome, see Trisomy 13 Pearson syndrome clinical features 48, 50 gene mutations 48, 49 PEO, see Progressive external ophthalmoplegia Perlman syndrome clinical features 57 genetics 57, 58 PIT-1, see POU1F1 Pituitary organogenesis 83, 84 posterior lobe development abnormalities 84, 85 ectopic posterior pituitary 85–92 endocrine consequences of disorders 90, 91 phenotypes of developmental syndromes 86 transcription factors, see HESX1, LHX3, LHX4, POU1F1, PROP1, SOX2, SOX3 PKS, see Pallister-Killian syndrome POLG, mutation in mitochondrial diseases 47, 48 POLIP, clinical features 44 POU1F1 genotype-phenotype correlations 77 hypopituitarism role 67, 76–78 mouse mutants 76

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Prader-Willi syndrome (PWS) clinical features 23–25, 119 epidemiology 23 etiology 119 genetic counseling 27 growth 119 growth hormone secretion and therapy 24, 25, 120 imprinting regulation 22–26 Progressive external ophthalmoplegia (PEO) clinical features 42, 43 gene mutations 41 PROP1 Ames dwarf mouse mutant 74, 75 genotype-phenotype correlations 77 hypopituitarism role 67, 75, 76, 78 recessive inheritance of mutations 75 Pseudoachondroplasia clinical features 128 etiology 128 growth 128 growth hormone secretion and therapy 129 PTEN, Bannayan-Riley-Ruvalcaba syndrome mutations 58 PWS, see Prader-Willi syndrome RSPO1, mutations in disorders of sexual development 178 SCO1, mutation in mitochondrial diseases 46 Septo-optic dysplasia (SOD), HESX1 mutations 68 Sexual development, see Cryptorchidism, Disorders of sexual development SGBS, see Simpson-Golabi-Behmel syndrome SHANK3, deletion 59 Silver-Russell syndrome (SRS) chromosomal aberrations chromosome 7 12, 13 chromosome 11 14, 15 chromosome 15 11, 12 chromosome 17 11, 12 clinical features 11, 123 genetics 11, 123 genotype-phenotype correlations 17, 18 growth 123, 124 growth hormone secretion and therapy 124, 125 imprinted gene networks 16, 17 Simpson-Golabi-Behmel syndrome (SGBS) clinical features 55, 101

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gene mutations 55 hyperinsulinemic hypoglycemia 101 SLOS, see Smith-Lemli-Opitz syndrome Smith-Lemli-Opitz syndrome (SLOS), gene mutations 179 SOD, see Septo-optic dysplasia Sotos syndrome clinical features 56, 100 gene mutations 56, 100 hyperinsulinemic hypoglycemia 100 SOX2 developmental expression 71 genotype-phenotype correlations 77 hypopituitarism role 67, 71, 72 SOX3 alanine expansions 71 duplication 70 ectopic posterior pituitary role 89 genotype-phenotype correlations 77 hypopituitarism role 67, 70, 71 knockout moue 69, 70 X-linked mental retardation mutations 69 SOX9, mutations in disorders of sexual development 176 SRS, see Silver-Russell syndrome STAT5b, mutations 146, 147 SURF-I, deficiency 40 Timothy syndrome clinical features 104 gene mutations 105 hyperinsulinemic hypoglycemia 105 TK2, mutation in mitochondrial diseases 46, 47 Trisomy 13 clinical features 103 hyperinsulinemic hypoglycemia 103 Turner syndrome clinical features 103, 104, 115 etiology 115 growth 115 growth hormone secretion and therapy 116, 117 hyperinsulinemic hypoglycemia 103, 104 22q13 deletion syndrome clinical features 58, 59 gene mutations 59 UBE3A, Angelman syndrome and loss of function 22 Ucx4.1, null mutation 85

Subject Index

Uniparental disomy (UPD), Silver-Russell syndrome 13 Angelman syndrome 26 Prader-Willi syndrome 25 UPD, see Uniparental disomy Usher syndrome clinical features 105 hyperinsulinemic hypoglycemia 105, 106

Subject Index

Weaver syndrome clinical features 57 gene mutations 57 Wilms’ tumor, imprinting center 1 absence of microdeletions 7, 8 WNT4, mutations in disorders of sexual development 178 WT1, mutations in disorders of sexual development 176

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